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What is the translation termination efficiency in mammalian cells?

What is the translation termination efficiency in mammalian cells?


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When I express proteins in bacteria I put at least two stop codons at the end of the gene to increase the termination efficiency. Is this the case in eukaryotic cells too? If I put a single stop codon is there a risk for the ribosomal complex to readthrough and continue translation?


There is a paper for transfection of mammalian cells here which has a bit of comparison of stop codon and protein yield.

Otherwise this article suggests having a 'rare' codon after the stop to prevent readthrough. But yes, I'm not sure why one wouldn't simply put two stops after each other.


UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals

Translation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.

Results

We have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10 -4 . We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon. There was no effect of amino acid identity or codon frequency. However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels. This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA. We then examined the importance of the downstream context using eight other constructs. No direct correlation between the +6 nucleotide and readthrough efficiency was observed.

Conclusions

We conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery. Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.


Termination-reinitiation occurs in the translation of mammalian cell mRNAs.

Many examples of internal translation initiation in eucaryotes have accumulated in recent years. In many cases terminators of upstream reading frames precede the internal initiation site, suggesting that translational reinitiation may be a mechanism for initiation at internal AUGs. To test this idea, a series of recombinants was constructed in the mammalian expression vector pSV2. Each contained a dicistronic transcription unit comprising the coding sequence for mouse dihydrofolate reductase (DHFR) followed by the gene for xanthine-guanine phosphoribosyl transferase (XGPRT) from Escherichia coli. Various versions of this pSV2dhfr-gpt recombinant plasmid altered the location at which the DHFR reading frame was terminated relative to the XGPRT initiation codon and demonstrated that this is a critical factor for the expression of XGPRT activity in transfected Cos-1 cells. Thus, when the DHFR frame terminated upstream or a very short distance downstream of the XGPRT initiator AUG, substantial levels of XGPRT activity were observed. When the DHFR frame terminated 50 nucleotides beyond the XGPRT initiator, activity was reduced about twofold. However, when the DHFR and XGPRT sequences were fused in-frame so that ribosomes which initiated at the DHFR AUG did not terminate until they encountered the XGPRT terminator, production of XGPRT activity was abolished. This dependence of internal translation initiation on the position of terminators of the upstream reading frame is consistent with the hypothesis that mammalian ribosomes are capable of translational reinitiation.


Abstract

Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control of gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions in ribosomal initiation complexes, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity (which affects almost all scanning-dependent initiation) and through sequence-specific RNA-binding proteins and microRNAs (which affect individual mRNAs).


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Materials and methods

Plasmid constructions

The TPI gene was mutagenized ( Kunkel et al., 1987 ) within a 420 bp BamHI–SmaI fragment that includes sequences from exon 1 and intron 1 and that was propagated as a single strand in pGEM7Zf(+) DNA. Nonsense mutations within codons 1, 2 and 10 were introduced using the antisense mutagenic primers 5′-CCTGGAGGG CTA CATGGCCGAG-3′, 5′-GAACTTCCTGGA CTA CGCCATGGCC-3′ and 5′-CTTCCAGTT TCA CCCAACG-3′ (in which the bold face nucleotides deviate from the corresponding TPI gene sequence and underlined nucleotides correspond to the altered codon). Additionally, codon 14 was changed from encoding methionine to encoding valine using the antisense mutagenic primer 5′-TTCCGCCCGTT AAC CTTCCAGTTTC-3′, either in a nonsense-free context or in the context of a nonsense codon at position 1, 2 or 10. 0Met→Val was generated using the antisense mutagenic primer 5′-GGAGGGCGC AAC GGCGCTGGAG-3′. 14Met out-of-frame was generated using the antisense mutagenic primer 5′-CTGCTTCC GG GCCCGTTCATCTTCCAGT C T-3′, which inserted a G nucleotide within codon 11 and a CC dinucleotide within codon 17. 14Met→Val out-of-frame was generated from 14Met out-of-frame using 5′-CCGGGCCCGTT AAC CTTCCAGTCTTC-3′.

After DNA sequencing to confirm the mutations had been introduced, pMT-TPI DNAs ( Cheng and Maquat, 1993 ) harboring each of the mutations and pSP6-TPI–CAT DNAs harboring some of the mutations were constructed by fragment substitution. The fragment substituted in constructing the pMT-TPI derivatives was BamHI–SmaI. The fragment substituted in constructing the pSP6-TPI–CAT derivatives was KpnI–EagI (see below). pSP6-TPI–CAT was constructed in several steps so that the CAT open translational reading frame began with codon 0Met of TPI and included the first 37 amino acids of TPI. First, the 270 bp PstI–EcoRI fragment from pCAT-Basic Vector (Promega), which includes polylinker sequences and the 5′-end of the CAT gene, was substituted for the 2.26 kbp PstI–EcoRI fragment, which extends from TPI intron 4 into 3′ flanking DNA. Next, the 1.28 kbp SacII–PstI fragment that spans TPI intron 1 to intron 4 was deleted, and the 1.11 kbp BamHI–EcoRI fragment that resides immediately downstream of the MT promoter and terminates at the EcoRI site of the CAT gene was inserted into the BamHI–EcoRI sites of pGEM7Zf(+). Subsequently, 750 bp that include TPI codon 38 to the SacII site of intron 1 plus 57 bp of the pCAT-Basic Vector polylinker and codon 0Met of CAT were deleted using the antisense mutagenic oligonucleotide 5′-GTATATCCAGTGAATTTTTTTCTC/GGTGTCGGCCGGCACC-3′ (where the slash specifies the site of the deletion). Finally, the mutagenized 364 bp BamHI–EcoRI fragment was inserted into the 2.61 kbp BamHI–EcoRI vector fragment of pMT-TPI, and the 750 bp BamHI–BamHI MT promoter fragment plus the 1.39 kbp EcoRI–EcoRI fragment that extends from the CAT reading frame into 3′ flanking DNA were inserted at the BamHI and EcoRI sites, respectively, to generate pMT-TPI Norm –CAT. pMT-TPI 1Ter –CAT, pMT-TPI 2Ter –CAT, pMT-TPI 23Ter –CAT, pMT-TPI Norm, 14Met→Val –CAT, pMT-TPI 1Ter, 14Met→Val –CAT and pMT-TPI 2Ter, 14Met→Val –CAT were constructed by substituting the 784 bp KpnI–EagI fragment, which extends from within the MT promoter to TPI exon 1, with the corresponding nonsense fragment. Subsequently, pSP6-TPI–CAT derivatives were constructed by inserting the 1.74 kbp BamHI–BamHI fragment, extending from the 5′-untranslated region of the TPI gene into SV40 DNA, into the BamHI site of pGEM7Zf(+), downstream of the SP6 promoter.

Cell transfections and RNA purification

Mouse Ltk − cells were grown in minimal essential medium α containing 10% fetal bovine calf serum and 5% bovine calf serum. Cells (3×10 7 /15 cm diameter dish) were transiently transfected with a test pMT-TPI plasmid (10 μg) and the reference pMT-G1 plasmid (10 μg) by using DEAE-dextran ( Cheng and Maquat, 1993 ). After 48 h, cells were harvested. Total L cell RNA was isolated using guanidine isothiocyanate and cesium chloride gradient centrifugation ( Cheng and Maquat, 1993 ). Nuclear and cytoplasmic RNA was isolated using Method 1 of Belgrader et al. (1994) .

RNA blot hybridization

Total, nuclear or cytoplasmic cell RNA (25 μg) was electrophoresed in a 1.5% agarose gel, transferred to a nylon membrane and hybridized to a 299 bp NdeI–NcoI fragment that derives from the 3′-untranslated region of human TPI cDNA and a 170 bp BalI–DraI fragment from the mouse β major -globin gene that consists of 158 bp of exon 3 plus 3′ flanking sequences ( Cheng and Maquat, 1993 ). Prior to hybridization, each fragment was 32 P-labeled by random priming ( Fineberg and Vogelstein, 1983 ). Hybridized radioactivity was quantitated using a PhosphorImager (Molecular Dynamics) and visualized using autoradiography.

Coupled transcription–translation of pSP6-TPI–CAT constructs in vitro

Test pSP6-TPI–CAT plasmid (1 μg) and reference luciferase control DNA (0.25 μg Promega) were transcribed from the SP6 promoter, and product RNA was translated using 25 μl of the TNT Reticulocyte Lysate System (Promega) that couples transcription by SP6 RNA polymerase and translation of the resulting RNA. A portion (6 μl) of the reaction mixture was brought to pH 5 using 1 μl of 1 M acetic acid, incubated on ice for 30 min and then centrifuged at 12 000 g and 4°C for 15 min in order to remove hemoglobin ( Shaun et al., 1984 ), which co-migrates with TPI–CAT protein. Pellets were dissolved in 50 μl of 1× SDS sample buffer (Promega), and 10 μl were denatured at 80°C and electrophoresed in a 12% acrylamide gel. 35 S-Labeled TPI–CAT and luciferase proteins were quantitated using a PhosphorImager and visualized using autoradiography.


DISCUSSION

Stop signal: a sequence element with multifaceted contributions to efficiency

A number of studies have provided evidence that nucleotides downstream of the eukaryotic translational stop codon can affect the efficiency of translation termination ( 21, 28, 30–32). While nucleotides close to the codon could have a direct effect by interacting with the decoding release factor, eRF1, as shown with the +4 base ( 6, 7) it has been puzzling how nucleotides quite distant could have such a direct effect. In the current study, we have investigated the influences of those nucleotides within the mRNA channel of the ribosome, when a stop codon is in the A site of the decoding centre, during translation of that codon.

Cryo-EM studies of the eukaryotic translational termination complex with eRF1 have shown that the +4 and +5 nucleotides of the stop codon context base stack with 18S rRNA bases G626 and C1698 respectively ( 6–8). Stacking would be more stable if a purine were present in the +4 position (G626) ( 6), and a purine in the +5 position (C1698) ( 8) increasing the stability of the decoding complex. Additionally, hydroxylation of a proline residue in the ribosomal protein uS12, which contacts the mRNA backbone near the +4 nucleotide, modulates readthrough efficiency in a direction dependent on the base in the +4 position, implying a special contribution of this nucleotide to the termination process ( 67). Consistent with this we found that the position following the A site stop codon (+4) has a particularly strong influence on the efficiency and fidelity of the decoding event. Additionally, the next two positions (+5 and +6) are also significantly influential in a complex manner, both from the perspective of the identity of the nucleotide in each position and through combinations of nucleotides from +4 to +6. This indicates that the interaction between the mRNA and the ribosomal components, like the rRNA nucleotides that line the channel, influence the translation decoding rate. RNase footprinting studies indicate that the length of mRNA protected within the eukaryotic ribosome is approximately 28–31 nt, depending on digestion conditions ( 60, 61, 68, 69). This suggests that the mRNA channel in the eukaryotic ribosome protects a very similar number of nucleotides to that of the bacterial ribosome ( 59, 70). Consistent with this we found that even in the outer reaches of the channel near to the entry point for the mRNA, the +8 nucleotide position can have strong effects to improve the efficiency of a poorly performing +1 UGA +3 stop codon as an efficient termination signal. Although we conducted only limited studies of sequence variations for the +7 to +9 distant positions, the results suggested the +8 position was dominant among this trio, with +7 and +9 not particularly influential in establishing an effector pattern (Figure 4). A similar complex pattern was observed in the readthrough element of tobacco mosaic virus. Notably, however, the +9 nucleotide has been observed to have a significant effect on readthrough depending upon the upstream context, indicating that caution is required when generalising these results to all stop signals ( 30, 31).

Both bioinformatics (Figure 5) and previous in vitro and in vivo experimental studies have implied the existence of a eukaryotic translation termination element that encompasses nucleotides both upstream and downstream of the stop codon ( 20, 22, 35, 45, 71). Parts of such an indicative element could have quite different influences on the efficiency of termination depending on where they are positioned. For example, the upstream sequence through its coding potential determines which amino acids are present in the PTC and exit tunnel as well as which tRNAs are favoured in the E and P sites preceding a stop codon. A nascent peptide designated MTO1 within the Arabidopsis thaliana CGS1 coding region causes ribosomes to stall in response to S-adenosyl-l-methionine during elongation ( 72). Expression of the human cytomegalovirus (CMV) gp48 gene is reduced by translation of its uORF2 nascent peptide ( 73), which causes ribosomes to stall at the uORF2 termination codon ( 74). These studies reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.

Eukaryotic eRF3 strongly stimulates peptide release by eRF1 ( 75) through its GTPase activity indicating a role for class-2 release factors in eukaryotic termination. It is of note that binding of eRF1–eRF3–GTP to pre-termination complexes (pre-TCs) also induces a 2-nt forwardshift of their toeprint ( 75), caused by the compaction of the mRNA in the termination complex ( 6, 76). Therefore, it is possible that sequences particularly amenable to such contraction would be favoured, suggesting another mechanism of how the extended termination signal could operate in vivo.

It has emerged that the evolutionarily distinct bacterial and eukaryotic decoding release factors interact with the stop codon through different parts of their structures, including the major recognition loop and helix a5 region for bacterial RFs ( 77, 78) and three distinctly separate peptide regions from the N-domain of eRF1 that surround the stop codon on the ribosome ( 5, 6). The exquisite geometry required for productive interaction and recognition of the stop codons highlights the importance of the correct orientation of the bases with respect to the amino acids of the eRF1. There is strong evidence for a significant conformational change in eRF1 on binding to eRF3 in solution or alone on the ribosome, causing eRF1 to adopt a bent conformation that resembles a tRNA ( 5–8, 79, 80). This dynamic structural change highlights how each individual factor/nucleotide interaction might be highly sensitive to perturbation from the distal nucleotides. As the more distal downstream bases in the mRNA can make interactions with ribosomal moieties in the channel they may distort the positioning of the upstream RNA bases affecting individual factor/nucleotide interaction required for decoding.

The overall conformation of the ribosome in the pre-termination state resembles the post-translocation state ribosome, with peptidyl-tRNA in the P site, which is locked in the nonratcheted conformation ( 7). In this conformation, there is a ∼8.5° rotation of the small subunit compared to the ratcheted state, reminiscent of the range of rotation for bacterial ribosomes ( 70, 81). Therefore, during translation termination, with sense codons in the P and E sites, there is a closure of the ribosomal mRNA channel that must trigger a network of interactions between the mRNA and the ribosome. This could explain why the downstream nucleotides distant from the stop codon can influence termination so markedly. In the model generated from the cryo-electron microscopy structure of a termination complex reported by Shao et al. ( 8) continuous density around the +7 base is observed which is perhaps indicative of direct contact between the ribosome and mRNA in the entrance channel.

The translation termination elements of UAA and UAG

UAA and UAG stop codons have a very high degree of termination fidelity and competitiveness against near-cognate events. It is tempting to dismiss the concept of a signal element with these codons since they are less markedly influenced by the downstream nucleotides. The oligonucleotide UAAC is, however, several-fold less efficient than UAAG at facilitating termination in a cell-free in vitro termination assay where there is no competition from tRNAs to decode the signal and the UAA codon itself is inactive ( 65). In our current study in cultured cells, the identity of the +4 nucleotide also has a significant effect on fidelity, so evidence for a four-base stop signal is compelling even with these high strength signals.

The patterns of stop failure were different between UAA and UAG signals, possibly resulting from the differences in UAG stop codon recognition by eRF1 (utilising E55) from that of UAA ( 6). There was, by contrast, a good correlation in the patterns for particular signals where data are available between yeast and our two mammalian cell lines, consistent with the high conservation of eRF1 among eukaryotes ( 82). The twelve sequences investigated by Bonetti et al. ( 21) to examine the effect of the +4 base on readthrough in yeast were included in our examined sequences, and comparison between the two studies reveals a comparable pattern of termination failure rates for the UAGNAU and UAANAU signals. However, there is a difference in signal readthrough in the UAGNAU context, with our study identifying higher readthrough with the UAGUAU context than Bonetti et al., who reported higher readthrough with UAGCAU ( 21). These differences may reflect the reported influence of the last C terminal amino acid and mRNA sequence 5′ of the stop codon on termination efficiencies ( 28, 29), as the 5′ context varied significantly between the two studies.

The translation termination element of UGA

Accurate decoding of the UGA stop codon was strongly dependent upon the three downstream nucleotides. The most marked example is with C in the +4 position immediately following the stop codon, which results in termination failure up to six-fold above the median value for the 64 contexts examined (Figure 1B). These results correlate with structural studies that show stacking between the +4 nucleotide and residue G626 of 18S rRNA would be more stable with purines in this position ( 6). This nucleotide stacking might increase termination efficiency through stabilising the interaction with eRF1 and the mRNA to contribute significant interaction energy. This observation correlates well with bioinformatic analyses that have found that the termination sequences TGAC and TGAT are under-represented in eukaryotic genomes ( 45). Stop codon recognition has been proposed to occur in two stages, an initial recognition, followed by a tightening of the interaction after GTP hydrolysis ( 83). The second step has been proposed to occur more slowly for UGA codons, and this would allow a greater chance of a near-cognate decoding event to compete. Additionally, as UGA stop codons make unique interactions with eRF1 ( 6), it is possible that these accommodations also affect the rate of decoding. It is significant that higher concentrations of eRF1 and presumably higher probability of occupancy of the A site enhanced the chance of a productive termination event at weaker UGA contexts ( 84).

The amelioration of nonsense mutations in inherited diseases that promote premature translational termination has been of continuing clinical interest. Stop codon failure at premature termination codons, influenced by context, may highlight sites at which the nonsense mutation may be more amenable to readthrough manipulation. Also, since premature termination triggers nonsense-mediated mRNA decay (NMD) ( 85) an extended termination signal may be important within the cell for translational machinery to distinguish between mature and premature stop codons ( 39).

An increasing number of highly efficient ‘programmed’ readthrough elements have been described in eukaryotes, sometimes with a defined biological function for the extended, ‘read-through’ polypeptides ( 86–89). For example, in Drosophila spp. it has recently been reported that an evolutionarily recent point mutation causing a premature termination codon can facilitate readthrough approaching 100% efficiency, resulting in the translation of a functional protein from an apparent pseudogene. Although the extended sequence that allows this to occur is not understood, the effect is independent of the identity of the stop codon but strongly dependent upon the +4 nucleotide ( 90). Furthermore, a case report of a patient with a predicted premature termination codon but with a weak termination context— +1 UGACUA +6 , among the weakest observed in our library—that can facilitate readthrough to mitigate the expected fatal disease phenotype has been reported ( 35). Although the nucleotides directly upstream and downstream of the described endogenous readthrough-prone termination contexts are frequently described to affect termination, in many instances an extended signal, often containing a structured element, is required to mediate efficient readthrough ( 91).

The biological function of the translation termination sequence element

A key question is whether the influence of the downstream sequence on the efficiency of stop codon decoding, as we have shown, is simply variation within an allowable physiological window, or whether there is a broader significance for the overall biology of the eukaryotic cell. In prokaryotes, where transcription and translation are spatially linked, efficient termination signals might convey a selective advantage to the organism and hence be positively selected over time. Indeed, in Escherichia coli the most highly expressed genes have the most rapidly decoded stop signals (predominantly UAAU) ( 92). What happens in eukaryotes, where transcription and translation are separated spatially? One key feature may lie with the role the translation termination sequence plays in the proposed mRNA recycling loop with eRF1 and eRF3 ( 93). More efficient sequence elements will transit eRF1 and eRF3 more rapidly, expediting the formation of the recycling loop and maintaining mRNA stability, increasing the rate of translation and the amount of protein expressed per transcript.

Do relatively inefficient termination signals also have a specific function? The ability of specific sequences to promote rare recoding events like stop codon readthrough (e.g. the histidine decarboxylase gene), selenocysteine incorporation at a UGAC (e.g. the glutathione peroxidase gene) and frameshifting also at a UGAC (e.g. the antizyme gene) allow for expression of a small alternative transcriptome (reviewed in ( 94)). Also, human genes that contain inefficient termination signals have common biological functions, such as cell cycling and anti-apoptosis ( 95). This is an intriguing observation considering that cell cycling proteins interact with eRF3 ( 96), over-expression of eRF1 and eRF3 in some cell lines causes apoptosis (Cridge, unpublished), and overexpression of eRF3/GSPT1 has been reported in intestinal type gastric tumors ( 97). Is there a hidden code in the nucleotides surrounding the stop codon that our study has highlighted, providing a subtle layer of regulation for the expression of a gene or co-ordinated pathways of genes, only the most obvious of which have so far been revealed?


Nonsense-mediated mRNA decay in mammals

Nonsense-mediated mRNA decay (NMD) in mammalian cells generally degrades mRNAs that terminate translation more than 50-55 nucleotides upstream of a splicing-generated exon-exon junction (reviewed in Maquat, 2004a Nagy and Maquat, 1998). Notably, dependence on exon-exon junctions distinguishes NMD in mammalian cells from NMD in all other organisms that have been examined, including Saccharomyces cerevisiae and Drosophila melanogaster (reviewed in Maquat, 2004b). NMD downregulates spliced mRNAs that prematurely terminate translation so production of the potentially toxic truncated proteins that they encode. NMD also downregulates naturally occurring mRNAs, such as an estimated one-third of alternatively spliced mRNAs, certain selenoprotein mRNAs, some mRNAs that have upstream open reading frames, and some mRNAs that contain an intron within the 3′ untranslated region (Hillman et al., 2004 Mendell et al., 2004 Moriarty et al., 1998). In fact, it is thought that NMD has been maintained throughout evolution not only because it degrades transcripts that are the consequence of routine abnormalities in gene expression but also because it is widely used to achieve proper levels of gene expression. Although disease-associated mutations that result in the premature termination of translation led to the discovery of NMD, it is not likely that this type of mutation ever drove significant evolutionary selection. Nevertheless, some of these mutations nicely illustrate the importance of NMD. For example, nonsense mutations within the last exon of the human β-globin gene do not elicit NMD because there is no downstream exon-exon junction. As a consequence, the resulting truncated β-globin has near-normal abundance, fails to properly associate with α-globin and causes a dominantly inherited form of what is otherwise (e.g. for nonsense codons located within exons other than the last exon) a recessively inherited thalassemia (Thein, 2004).FIG1

The importance of NMD is exemplified by the findings that mouse embryos that cannot perform NMD because they lack a key NMD protein, Upf1, resorb shortly after implantation (Medghalchi et al., 2001). Furthermore, blastocysts that have the same defect, isolated 3.5 days post-coitum, undergo apoptosis in culture after a brief growth period (Medghalchi et al., 2001). The inviability of NMD-deficient embryos and cells probably reflects the combined failure to regulate natural substrates properly and eliminate transcripts that were generated in error. Note that, Upf1 has been shown to function in other pathways, as well as NMD (see below), which may also contribute to the observed inviability.

NMD in mammalian cells is a consequence of a pioneer round of translation (Chiu et al., 2004 Ishigaki et al., 2001 Lejeune et al., 2004). As illustrated in the poster, precursor (pre)-mRNA in the nucleus is bound to by the major nuclear cap-binding protein (CBP) CBP80-CBP20 heterodimer and, after 3′-end formation, the major nuclear poly(A)-binding protein (PABP) PABPN1 (Chiu et al., 2004 Ishigaki et al., 2001). Pre-mRNA splicing generates spliced mRNA that is bound by CBP80, CBP20, PABPN1 and the major cytoplasmic PABPC (Chiu et al., 2004 Ishigaki et al., 2001 Lejeune et al., 2004) as well as an exon junction complex (EJC) of proteins that is deposited, as a consequence of splicing, ∼20-24 nucleotides upstream of each exon-exon junction (Le Hir et al., 2000a Le Hir et al., 2000b). Constituents of EJCs include Y14, RNPS1, SRm160, REF/Aly, UAP56, Magoh, Pnn/DRS, eIF4AIII, PYM and Barentsz/MLN51 (Bono et al., 2004 Chan et al., 2004 Custodio et al., 2004 Degot et al., 2004 Ferraiuolo et al., 2004 Kataoka et al., 2000 Kim et al., 2001 Le Hir et al., 2001 Le Hir et al., 2000a Le Hir et al., 2000b Lejeune et al., 2002 Li et al., 2003 Luo et al., 2001 Palacios et al., 2004 Shibuya et al., 2004). The EJC also contains additional proteins, including the NMD factors Upf3 (also called Upf3a) or Upf3X (also called Upf3b), Upf2 and, presumably transiently, Upf1 (Kim et al., 2004 Lykke-Andersen et al., 2000 Lykke-Andersen et al., 2001 Mendell et al., 2000 Ohnishi et al., 2003 Serin et al., 2001). Either Upf3 or Upf3X, each of which is mostly nuclear but shuttles to the cytoplasm and interacts with Upf2, is thought to recruit Upf2, which concentrates along the cytoplasmic side of the nuclear envelope (Kadlec et al., 2004 Lykke-Andersen et al., 2000 Serin et al., 2001).

The resulting mRNP constitutes the pioneer translation initiation complex (Chiu et al., 2004 Ishigaki et al., 2001 Lejeune et al., 2002 Lejeune et al., 2004). This complex is thought to undergo a `pioneer' round of translation either in association with nuclei, in the case of mRNAs that are subject to nucleus-associated NMD, or in the cytoplasm, in the case of mRNAs that are subject to cytoplasmic NMD. If NMD occurs, it is the consequence of nonsense codon (NC) recognition during this pioneer round of translation (Chiu et al., 2004 Ishigaki et al., 2001 Lejeune et al., 2004). Upf1 may function as a component of the translation termination complex before it functions in NMD, considering that NMD requires translation termination and Upf1 associates with eukaryotic translation release factors 1 (F. Lejeune and L.E.M., unpublished) and 3 (G. Singh and J. Lykke-Andersen, personal communication). Upf1 might associate with mRNA regardless of whether termination occurs at a position that elicits NMD. If translation terminates at an NC that resides more than 50-55 nucleotides upstream of an exon-exon junction, then Upf1 is thought to elicit NMD by interacting with EJC-associated Upf2 (Maquat, 2004a Lykke-Andersen et al., 2000 Mendell et al., 2000 Serin et al., 2001). Consistent with a role for EJCs in NMD is the observation that NC-containing mRNAs that derive from intronless genes fail to undergo NMD (Brocke et al., 2002 Maquat and Li, 2001).

Once the mRNA is remodeled so that eukaryotic translation initiation factor (eIF)4E replaces CBP80-CBP20 at the mRNA cap, PABPC replaces PABPN1 at the poly(A) tail, and EJCs have been removed from mRNA, the mRNA becomes immune to NMD (Chiu et al., 2004 Ishigaki et al., 2001 Lejeune et al., 2002). Translation has been reported to remove Y14 (Dostie and Dreyfuss, 2002), and it may remove other mRNA-binding proteins as well. Although these conclusions derive largely from studies of mRNP structure, they are consistent with kinetic analyses indicating that NMD is restricted to newly synthesized mRNA and does not detectably target steady-state mRNA (Belgrader et al., 1994 Cheng and Maquat, 1993 Lejeune et al., 2003).

Cell fractionation studies indicate that most nonsense-containing mRNAs are subject to nucleus-associated NMD (reviewed in Maquat, 2004a). This means that mRNA decay occurs prior to the release of newly synthesized mRNAs into the cytoplasm. Nucleus-associated NMD has been proposed to occur within the nucleoplasm, but it is generally thought to take place during or after mRNA transport across the nuclear pore complex (Dahlberg et al., 2003 Maquat, 2002). A fraction of mRNAs is subject to cytoplasmic NMD (e.g. Dreumont et al., 2004 Moriarty et al., 1998). What destines some mRNAs for nucleus-associated NMD and others for cytoplasmic NMD is currently unknown.

NMD in mammalian cells occurs both 5′-to-3′ and 3′-to-5′ it thus involves decapping and 5′-to-3′ exonucleolytic activities as well as deadenylating and 3′-to-5′ exosomal activities (Lejeune et al., 2003 Chen and Shyu, 2003). It remains to be determined whether NMD occurs in association with translating ribosomes or so-called cytoplasmic foci, which appear to be ribosome-free sites of general mRNA decay (Cougot et al., 2004 Ingelfinger et al., 2002). Notably, the efficiency of NMD in mammalian cells is generally not influenced by NC position, indicating that a higher number of downstream EJCs does not lead to more efficient NMD. However, NMD can be augmented by additional mechanisms that are not well understood. For example, replacing exons 2-4 and flanking intron sequences of the triosephosphate isomerase (TPI) gene with the 383-bp VDJ exon and flanking intron sequences of the T-cell receptor β (TCR-β) gene, which generates mRNA that is more efficiently targeted for NMD than TPI mRNA, increases the efficiency with which TPI mRNA undergoes NMD >15-fold (Gudikote and Wilkinson, 2002). The efficiency of NMD is increased only when the TCR-β sequence is located upstream of an NC.

An understanding of how various factors function in NMD is far from complete. Upf1 is an ATP-dependent group 1 RNA helicase and phosphoprotein (Bhattacharya et al., 2000 Pal et al., 2001 Sun et al., 1998) that, as described above, presumably triggers NMD by interacting with Upf2 at an EJC that resides sufficiently far downstream of an NC. Also, as noted above, Upf2 and either Upf3 or Upf3X, which appear to have distinct but overlapping functions (Lykke-Andersen et al., 2000 Serin et al., 2001 Gehring et al., 2003), are components of the EJC. In fact, Upf3 and Upf3X consist of multiple isoforms that result from alternative pre-mRNA splicing. Whether or not Upf2, Upf3 and Upf3X are involved in Upf1 dephosphorylation, as are their orthologues in C. elegans (Page et al., 1999), remains to be determined. However, as in C. elegans (Grimson et al., 2004 Page et al., 1999), Upf1 phosphorylation is mediated by the PIK-related kinase SMG1 (Brumbaugh et al., 2004 Denning et al., 2001 Yamashita et al., 2001). Also as in C. elegans (Anders et al., 2003 Page et al., 1999) and, possibly, D. melanogaster (Gatfield et al., 2003), Upf1 dephosphorylation is mediated by SMG5 and, presumably, SMG6 and SMG7 (Chiu et al., 2003 Gatfield et al., 2003 Ohnishi et al., 2003).

Interestingly, factors that function in NMD have also been shown to function in other pathways. For example, SMG1 is an ATM-related kinase that is also involved in the recognition and/or repair of damaged DNA (Brumbaugh et al., 2004). SMG1 phosphorylates the tumor suppressor checkpoint protein p53 in response to UV and γ irradiation, and cells in which SMG1 has been downregulated accumulate spontaneous DNA damage and are sensitized to ionizing radiation (Brumbaugh et al., 2004). Providing another example, Upf1 is the δ helicase that partially co-purifies with DNA polymerase δ (Carastro et al., 2002). Upf1 (unlike Upf2, the only other NMD factor tested) also appears to function in nonsense-mediated altered splicing (NAS), a poorly understood pathway by which NCs influence the efficiency or accuracy of splicing (Mendell et al., 2002 Wang et al., 2002). In fact, Upf1 can be mutated so that it functions in NAS but not NMD (Mendell et al., 2002), indicating that the two pathways are genetically separable. Furthermore, Upf1 has recently been found to function in a new pathway called Staufen 1 (Stau1)-mediated mRNA decay (SMD) (Kim et al., 2005). In this pathway, the RNA-binding protein Stau1 interacts directly with Upf1 to elicit mRNA decay when bound sufficiently far downstream of an NC, including the normal termination codon. The results of microarray analyses indicate that there are a number of natural targets for SMD (Kim et al., 2005). Finally, Upf1 interacts with PABPC and forms distinct complexes of approximately 1.3 MDa and 400-600 kDa that appear to differ in their content (Schell et al., 2003). The functional significance of all these findings remains unknown. Multiple roles for NMD factors are also evident in the case of SMG5, SMG6 and SMG7, which are identical to the Ever Shorter Telomere (EST) proteins EST1B, EST1A and EST1C, respectively (Reichenbach et al., 2003 Snow et al., 2003). Each associates with active telomerase and is involved in telomere integrity (Reichenbach et al., 2003 Snow et al., 2003).

As is evident from this short overview, many mechanistic details of NMD still require resolution. In the future, it will also be important for us to understand the extent to which NMD regulates the level of proper mRNA production as opposed to degrading mRNAs that produce aberrant and, therefore, potentially harmful proteins. How NMD is mechanistically linked to other cellular processes, some of which can also be viewed as a type of quality control, requires further study.


Results

Pre-mRNA splicing enhances polysome association of mRNA

We previously reported that spliced mRNAs exhibit increased translational yield as compared with no-intron mRNAs in mammalian tissue culture cells (Nott et al. 2003). To determine if this effect was a consequence of increased translational efficiency, we analyzed the distribution of several spliced or no-intron mRNAs on cytoplasmic polyribosomes (polysomes Fig. 1). Two constructs, TCR-β and β-globin, contained two introns each (Fig. 1A,D Lykke-Andersen et al. 2000 Nott et al. 2003), whereas a third, Renilla luciferase, contained a single intron (Nott et al. 2003). Plasmids expressing these and their respective no-intron controls were transiently transfected into HeLa cells, and polysome analysis was performed 24 h later. Endogenous cyclophilin mRNA served as an internal control for relative polysome integrity. Treatment of the cells with puromycin, an antibiotic that causes nascent peptide release and polysome disruption, confirmed that sedimentation of the reporter mRNAs and cyclophilin in denser fractions did reflect polysome association (data not shown).

Splicing enhances mRNA polysome association. (A,D) Schematic representation of TCR-β and β-globin constructs. Boxes represent exons, and lines connecting them denote introns. RPA probes are indicated by heavy lines. (B,C,E,F) Sucrose gradient fractionation of cytoplasmic extracts from cells expressing no-intron or intron-containing versions of TCR-β and β-globin. RNA extracted from each fraction (see Materials and Methods) was subject to RPA with probe E or J (A,D) and a probe specific to endogenous cyclophilin mRNA. Protected fragments were separated on a 10% denaturing polyacrylamide gel. Relative absorbance at 254 nm is depicted by thin lines, and the percent total mRNA in each fraction is shown by the dashed lines (scale on right).

Like the endogenous cyclophilin mRNA, the bulk of spliced TCR-β mRNA cofractionated with polysomes (Fig. 1B, fractions 5–12). In contrast, a large fraction of the no-intron TCR-β mRNA was found at the top of the gradient in the region containing individual ribosomal subunits and 80S monosomes (Fig. 1C). Similar results were observed for the β-globin (Fig. 1E,F) and the Renilla luciferase constructs (data not shown). Thus, for three different genes with different introns, splicing correlates with enhanced mRNA polysome association.

The exon junction complex plays a role in translation enhancement

We next wanted to determine whether the enhanced translational yield of spliced mRNAs could be observed when splicing was uncoupled from other RNA processing events (e.g., transcription and polyadenylation) and to what extent it was attributable to EJC deposition. This was most easily addressable in the Xenopus oocyte system, in which we injected the Renilla luciferase pre-mRNAs schematized in Figure 2B. Pre-mRNAs were designed with a 38- or 17-nt 5′-exon plus the human triose phosphate isomerase intron 6 (TPI intron). Because the EJC is deposited at a fixed distance, 20–24 nt upstream of an exon–exon junction, the 38- and 17-nt exons are just long enough or too short, respectively, to accept an EJC (Le Hir et al. 2001). Uniformly radiolabeled and m 7 GpppG-capped transcripts were generated in vitro using DNA templates terminating 4 nt downstream from the first in-frame stop codon. Thus, none of the injected RNAs contained a poly(A) tail. The AUG start codon was located in the 5′-exon, and several in-frame stop codons in the intron ensured that functional luciferase could not be translated from the unspliced pre-mRNAs (38 + I and 17 + I). Coinjected controls included U6Δss snRNA (to control for nuclear injection and nuclear envelope integrity), human initiator methionyl-tRNA (which is rapidly exported via an export pathway distinct from mRNA Jarmolowski et al. 1994), and unlabeled Firefly luciferase mRNA to control for translational differences between oocytes. By following both subcellular RNA distribution and luciferase activity as a function of time, we obtained information on both RNA export efficiency and its subsequent translation in the cytoplasm.

EJC-dependent stimulation of translational yield in Xenopus oocytes. (A) Overview of experimental procedure. (B) Schematic representation of in vitro transcribed, radiolabeled RNAs injected into Xenopus oocytes. TPI intron 6 (thick line) and flanking TPI exons (gray and black boxes) and Renilla luciferase ORF (white box) are indicated. First exons were either 38 or 17 nt in length. (C) Denaturing polyacrylamide gel electrophoresis of RNA extracted from nucleus (n) and cytoplasm (c) at 5 min, 4 h, and 24 h post-injection. (Top panel) 6% gel for Renilla RNAs (bottom panel) identical samples loaded on a 10% gel to separate the U6Δss RNA and tRNA. (D) Translational efficiencies at 24 h post-injection relative to 17 no I mRNA. Translational efficiencies from two independent experiments were calculated by first normalizing Renilla luciferase activity to Firefly luciferase activity and then dividing by cytoplasmic mRNA levels error bars represent the range.

Immediately after injection (5-min time point), both precursor RNAs were found predominantly in the nuclear compartment (Fig. 2C). Examination of the 4- and 24-h time points revealed that both pre-mRNAs were spliced and the mRNAs exported with comparable efficiencies. As is often observed in oocyte experiments, a significant percentage of the injected pre-mRNAs was also exported (Fig. 2C Le Hir et al. 2001). This is likely caused by an inability of the endogenous splicing apparatus to capture and retain all of the injected pre-mRNA before it can escape to the cytoplasm. Nonetheless, the presence of cytoplasmic pre-mRNA was of little consequence for our analysis because the pre-mRNAs were incapable of generating functional luciferase (see above), and no detectable luciferase activity was produced when the pre-mRNAs were injected directly into the cytoplasm (data not shown).

Protein expression was determined by monitoring cytoplasmic luciferase activities. Whereas neither construct produced detectable activity at 4 h (data not shown), light production was readily measurable in both cases by 24 h. A quantitative analysis of the relative translational yields was obtained by normalizing luciferase activities to cytoplasmic mRNA levels (Fig. 2D). This revealed that the 38 + I mRNA yielded 3.3 times more luciferase activity per mRNA molecule than did the 17 + I mRNA. This difference was not due to the 5′-UTR length as the respective no-intron control mRNAs were translated with equal efficiency in parallel experiments (data not shown). Thus, we conclude that the observed effect of splicing on translational yield is caused by EJC deposition and is independent of transcription and polyadenylation.

Individual EJC proteins enhance protein expression in a tethering assay

Having demonstrated that EJC deposition can mediate splicing-dependent enhancement of translational yield in Xenopus oocytes, we next wanted to determine whether this activity could be assigned to one or more specific protein(s). Thus, we tested whether the intron-dependent enhancement in translational yield could be replicated by tethering individual proteins within the ORF of Renilla luciferase. An intronless Renilla luciferase reporter carrying six MS2-binding sites at the 5′-end of the ORF (5′-6bs/Renilla Fig. 3A) was cotransfected into HeLa cells along with constructs expressing individual EJC proteins as fusions with bacteriophage MS2 coat protein (see Materials and Methods). A plasmid encoding a no-intron Firefly luciferase gene was also cotransfected as a control for both luciferase activity measurements and mRNA comparisons. The MS2 coat protein alone served as the control for reporter expression in the absence of a fusion partner. Expression of all fusion proteins was confirmed by Western blotting (data not shown).

Effects of tethering individual EJC proteins inside the Renilla luciferase ORF. (A) Schematic representation of 5′-6bs/Renilla reporter construct and Firefly control. Six MS2-binding sites were inserted 49-nt downstream of the start codon to be in-frame with the Renilla luciferase gene. (B) Levels of 5′-6bs/Renilla reporter mRNA and luciferase activity normalized to Firefly control and represented relative to MS2 alone. The data shown are the average of three to four independent experiments error bars represent standard deviation. (C) Nuclear and cytoplasmic distribution of 5′-6bs/Renilla reporter and Firefly control mRNA in the presence of various MS2 fusion proteins. RNA was extracted from fractionated nuclear (n) and cytoplasmic (c) compartments and analyzed by RPA (top panel). The percent of total mRNA in the cytoplasm (lower panel).

The fusion proteins fell into four functional classes: (1) those that had little or no effect, (2) those that increased reporter mRNA abundance only, (3) those that increased mRNA translational yield only, and (4) one that increased both mRNA abundance and translational yield (Fig. 3B). Constituting the first class were the DEK and REF2-1 MS2-fusions. DEK had no effect on either mRNA abundance or luciferase activity, whereas tethering of REF2-1 only slightly increased both. In contrast, coexpression of MS2–SRm160 resulted in a threefold increase in Renilla luciferase activity. A concomitant threefold increase in 5′-6bs/Renilla mRNA abundance indicated that although tethered SRm160 can enhance mRNA levels, it had no detectable effect at the translational level. The third class consisted of Y14-MS2 and Magoh-MS2, both of which increased protein expression three- to fourfold without significantly affecting reporter mRNA abundance. Finally, coexpression of MS2–RNPS1 increased reporter mRNA abundance (∼fourfold) but enhanced luciferase expression to a much greater extent (∼14-fold). Thus, tethering RNPS1 to the reporter mRNA led to an ∼fourfold increase in mRNA levels and an ∼threefold increase in translational yield.

We next examined whether any of the fusion proteins altered the nucleocytoplasmic distribution of Renilla reporter mRNA. Comparison to the cotransfected Firefly control revealed that only MS2–DEK had any effect on reporter mRNA distribution, but this decrease in the proportion of mRNA that was cytoplasmic was minor at best (Fig. 3C). Because tethering of Y14, Magoh, or RNPS1 had no effect on the nucleocytoplasmic distribution of the Renilla reporter mRNA, the enhancements in translational yield produced by these proteins were not simply attributable to more efficient cytoplasmic localization of the message.

Taken together, the above results indicate that when tethered as MS2 fusions near the 5′-end of an ORF, individual EJC components can mediate significant enhancements of gene expression. The sole effect observed here of tethering SRm160 was to increase mRNA levels. Y14 and Magoh increased protein expression, mostly by increasing the amount of protein produced per mRNA. Only RNPS1 increased both mRNA levels and translational yield.

Tethered Upf proteins also enhance translational yield

Of all the EJC proteins tested here, the only ones that increased translational yield when tethered inside the Renilla luciferase ORF were RNPS1, Y14, and Magoh. Interestingly, all of these proteins were previously shown to trigger NMD when tethered downstream from a termination codon in β-globin mRNA (Lykke-Andersen et al. 2001 Fribourg et al. 2003 Gehring et al. 2003). In contrast, other EJC proteins that did not enhance translational yield in the experiments presented here also failed to trigger NMD (Lykke-Andersen et al. 2001). We next wanted to ask if the ability to trigger NMD generally correlates with the ability to enhance translational yield.

Three other proteins previously documented to trigger NMD when tethered downstream from the termination codon in β-globin mRNA are the NMD factors Upf1, Upf2, and Upf3b (Lykke-Andersen et al. 2000). To test the effects of tethering these proteins inside an ORF, plasmids encoding the appropriate MS2-fusion proteins were cotransfected into HeLa cells with the 5′-6bs/Renilla reporter and Firefly control as described above. A β-globin-6bs reporter carrying six MS2-binding sites downstream from the normal termination codon was also cotransfected to monitor NMD activation by the expressed proteins (Fig. 4A Lykke-Andersen et al. 2000). The MS2-binding sites in the β-globin-6bs reporter were identical to the MS2-binding sites in the 5′-6bs/Renilla mRNA they differed only in their position relative to the respective ORFs. The Y14, Magoh, and RNPS1 fusions were also tested in this set of experiments. MS2–SRm160 served as a negative control because it neither enhances translational yield (Fig. 3B) nor triggers NMD (Lykke-Andersen et al. 2001).

Upf1, Upf2, and Upf3b also enhance the translational yield of 5′-6bs/Renilla mRNA. (A) Schematic representation of 5′-6bs/Renilla and β-globin-6bs constructs carrying identical MS2-binding sites. RPA probes are indicated with thick lines. (B) RPA of 2 and 8 μg of total mRNA extracted from transfected cells. (C) β-globin-6bs mRNA levels in the presence of MS2-fusion proteins relative to MS2 alone. The data reflect the average of two independent experiments error bars represent the range. (D) Renilla reporter mRNA levels in the presence of MS2-fusion proteins relative to MS2 alone. The data reflect the average of two independent experiments error bars represent the range. (E) Relative translational yields of Renilla luciferase. Luciferase activities were normalized to mRNA levels and are represented relative to MS2 alone. The data reflect the average of three independent experiments errors were propagated using standard algorithms.

Consistent with previous reports (Lykke-Andersen et al. 2000, 2001 Fribourg et al. 2003 Gehring et al. 2003), tethering of Upf1, Upf2, Upf3b, Y14, Magoh, or RNPS1 downstream from the termination codon significantly reduced β-globin-6bs mRNA abundance (Fig. 4B,C). However, none of the Upf proteins had any significant effect on the coexpressed 5′-6bs/Renilla reporter (Fig. 4B,D). Thus, when tethered inside an ORF, the Upf proteins neither increase nor decrease mRNA levels. In contrast, both the RNPS1 and SRm160 MS2-fusions led to higher 5′-6bs/Renilla mRNA levels as was observed in Figure 3. MS2–SRm160 also yielded a slight increase in β-globin-6bs mRNA abundance, consistent with its ability to increase mRNA levels (Fig. 3), but not trigger NMD (Lykke-Andersen et al. 2001).

Whereas the Upf proteins failed to affect mRNA levels when tethered inside the Renilla ORF, they did enhance the amount of active luciferase produced per mRNA. When translational yields were calculated by normalizing the luciferase activity to mRNA levels, all three Upf proteins increased the translational output to extents indistinguishable from RNPS1, Y14, and Magoh (Fig. 4E). As was the case for the latter proteins, the increased translational yields mediated by the tethered Upf proteins were unlikely caused by enhanced mRNA export because the nucleocytoplasmic localization of the reporter mRNA was unchanged in the presence of the MS2–Upf proteins compared with MS2 alone (data not shown). Thus, all proteins tested to date that are capable of triggering NMD when tethered downstream of a termination codon also lead to increased translational yields when tethered inside an ORF.

Tethered EJC and Upf proteins promote mRNA polysome association

The data presented in Figure 1 demonstrated that the greater translational yields observed with spliced mRNAs correlated with their increased polysome association. To determine whether tethering of an EJC or Upf protein could replicate this effect, we examined the polysome distribution of the 5′-6bs/Renilla reporter mRNA in the absence or presence of coexpressed MS2 proteins. In the absence of any MS2 fusion (vector) or in the presence of MS2 alone (MS2), a large proportion of the 5′-6bs/Renilla mRNA was found in fractions that contained ribosomal subunits and monosomes, whereas only a small fraction of the reporter mRNA cosedimented with polysomes (Fig. 5A,D). Thus, expression of MS2 coat protein alone had no effect on polysome association of 5′-6bs/Renilla mRNA. In contrast, coexpression of the MS2–RNPS1 fusion significantly enhanced the fraction of 5′-6bs/Renilla mRNA cosedimenting with polysomes (Fig. 5A, lower panel Fig. 5D). Distributions of endogenous cyclophilin mRNA and cotransfected Firefly control (data not shown) were unaffected in all experiments. A control experiment using puromycin to disrupt polysomes confirmed that the sedimentation of 5′-6bs/Renilla mRNA in denser fractions in the presence of MS2–RNPS1 was, indeed, due to polysome association (Fig. 5B).

MS2-fusions of RNPS1, Y14. Magoh and Upf proteins enhance polysome association of Renilla reporter mRNA. (A) Sucrose gradient fractionation of cytoplasmic extracts from cells expressing 5′-6bs/Renilla reporter mRNA and individual MS2-fusion proteins (indicated on left). The absorbance profile is representative of all three gradients. RNA extracted from each fraction was subjected to RPA with probe C (Fig. 3A) and a probe specific to endogenous cyclophilin mRNA. Protected fragments were separated on a 10% denaturing polyacrylamide gel. (B) MS2–RNPS1 cotransfected cells were treated with Puromycin prior to lysis and fractionation. (C) Sucrose gradient fractionation of cytoplasmic extracts from cells expressing 5′-6bs/Renilla reporter mRNA plus MS2 alone, MS2–Y14, MS2–Magoh, MS2–Upf1, MS2–Upf2, or MS2–Upf3b. The absorbance profile is representative of all gradients. (D) Quantitative representation of 5′-6bs/Renilla mRNA distribution in polysome gradients in the presence of indicated MS2-fusion proteins. Relative mRNA levels in each fraction were calculated as a percent of the total, and amounts in fractions 1–4 and 5–12 were pooled to represent mRNAs cofractionating with monosomes and ribosomal subunits and polysomes, respectively. The data represent the averages of two independent experiments, and error bars reflect the range.

We also examined the polysome distribution of the 5′-6bs/Renilla reporter tethered to Y14, Magoh, Upf1, Upf2, or Upf3b (Fig. 5C). As observed with RNPS1 tethering of Y14, Magoh and all three Upf proteins shifted a greater percentage of reporter RNA into polysome-containing fractions (Fig. 5C), whereas distribution of endogenous cyclophilin mRNA was unaffected (data not shown). A quantitative measure of mRNA distributions upon tethering the respective MS2-fusion proteins was obtained by calculating the relative mRNA abundance in fractions 1–4 (monosomes and ribosomal subunits) and fractions 5–12 (polysomes Fig. 5D). Thus, the translational enhancement observed upon tethering of the Upf proteins presumably occurs via the same mechanism as that mediated by splicing (Fig. 1) and EJC deposition.


Acknowledgements

We thank Beatrice Dimitriades for the help with the cell culture and western blot and Ana Correia, Anastasiya Börsch, and the members of the Zavolan group for their input on the project. We thank Martin Spiess for the generous gift of the NIH-3T3 cell line and Yaron Shav-Tal at Bar-Ilan University, Israel, for the FUCCI plasmids. We also thank Verena Jäggin and Telma Lopes from the FACS facility at D-BSSE, ETH, for the assistance with cell sorting experiments Janine Bögli from the FACS facility at Biozentrum, University of Basel, for the help with cell sorting and flow cytometry experiments Philippe Demougin from the Genomics Facility Basel for the genomic library preparation and the sciCORE team for their maintenance of the HPC facility at the University of Basel.

Review history

The review history is available as Additional file 8.

Peer review information

Barbara Cheifet and Andrew Cosgrove were the primary editors of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.


DNA to RNA to Protein one last time

After translation is completed we have a protein which came from mRNA that was transcribed from DNA. This protein product will eventually be trafficked to the location where it does its job. More information on protein trafficking will be covered in chapter 9.

Figure 4-15 illustrates the flow of this information and how the size of the genetic product gets smaller and smaller after each step. It is important to note that nucleic acids are relatively large in comparison to the typical protein. It is a misconception to think proteins, because they do most of the functions of the cell, are larger than the DNA or RNA molecules that make them.

Overall, in eukaryotes, a large amount of DNA is transcribed into mRNA. The mRNA is processed to add a 5’-cap, remove introns, and add a poly-A tail. Removal of intronic sequences greatly reduces the size of the mature mRNA (remember introns are typically much larger than exons). The mature mRNA is exported to the cytoplasm from the nucleus. In the cytoplasm the mRNA is translated, but two regions of the mRNA, the 3’-UTR and 5’-UTR remain untranslated. This doesn’t mean these sequences aren’t important it has been noted these regions are critical for the process of export out of the nucleus, translation, and the mRNA’s stability. Only the region that incorporates the start and stop codons in the mRNA are translated. Overall this means the protein product is much smaller than the mRNA for multiple reasons: 1) there are regions of the mRNA that are not translated (mentioned above) 2) it takes three RNA nucleotides (a codon) to get one amino acid and 3) amino acids are typically about much smaller (fewer atoms) than a ribonucleotide. This size difference is roughly illustrated in Figure 4-15.

Figure 4-15: Flow of genetic information from DNA to protein. A gene is transcribed into mRNA, which is processed. Notice the relative size of the gene compared to the mature mRNA (not to scale). After the mRNA is processed it is much smaller than the pre-mRNA, which is smaller in size compared to the original DNA sequence. The protein created after translation in the cytoplasm is even smaller and finally the protein folds into its three-dimensional structure.


Watch the video: Προβλέψεις Λα Λίγκα: Ποιά ομάδα θα πάρει το Ισπανικό πρωτάθλημα φέτος; (May 2022).


Comments:

  1. Segenam

    thanks for the article ... added to the reader

  2. Kunsgnos

    I think, that is not present.



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