Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation |核心内容:
Discussion We have performed RNA sequencing analysis on mRNA isolated from different fractions of a polysome gradient in order to analyse the translation rate of individual mRNA variants. We find that many mRNAs are translationally regulated during differentiation of ESCs to NPCs. We have validated that a shift in ribosomal load has a consequence for protein production with Tchp. Tchp is translationally upregulated in NPCs resulting in an increase in protein levels. TCHP was originally described as a keratin filament binding protein and plays a role in ciliogenesis and centrosomal function [35, 36]. TCHP has no known role in the nervous system but given its translational upregulation it may be a regulator of early neural differentiation. We identified 31 genes expressing multiple variants that are translationally regulated in a variant specific manner on NPC differentiation. In these cases multiple variants are detected but only one is under translational control on differentiation. In a number of cases there was a switch in the variant that was highly translated so that while the absolute levels of the RNA variants may be the same, their different translation rates will result in the protein products being present at different levels in ESCs and NPCs. Interestingly the role of the majority of these variants is not known so it remains to be determined how this variant specific translation impacts on neural differentiation. The method of RNAseq analysis of polysomal fractions is different from the alternative approach of ribosomal footprinting which provides information on the mRNA sequence physically bound by ribosomes and is used to infer translation rate [32, 37]. Despite the high resolution of the method the footprint sequences do not provide information on UTRs. In addition, currently the technique is not able to distinguish between different splice variants. Polysome profiling coupled to RNAseq provides information on variant specific ribosomal load and enables the analysis of corresponding UTR sequences. Using polysome profiling coupled with RNAseq, we find that in ESCs ten percent of RNA isoforms display different translation rates. This correlates with differences in the UTR sequences which likely drive the altered translation rate. This suggests that the relative protein levels of the two isoforms will not correlate with their mRNA levels. Alternate UTRs can arise through a number of different mechanisms including alternate TSS, alternative splicing and alternative polyA site selection. Our datasets were mapped to the RefSeq genome which has annotations for promoter start sites and known alternative splicing events. As such, our studies have focused only on variants arising from alternative transcription start site selection and alternative splicing. Our analysis does not take into account any APA that may be occurring and it is possible that the variants identified in our studies may be further regulated by APA in different cell types. We find that in ESCs 10% of mRNAs with multiple variants display different translation rates for each variant inferred from their ribosomal load. This strongly correlates with the variants having different 5’ and 3’UTR sequences. This represents over 67 genes with different detectable isoforms in ESCs. This demonstrates the importance of analysing the translation rates of RNA variants when studying gene expression patterns. We confirmed the differential ribosomal load of five variants by PCR and performed luciferase assays to confirm that the regulation was mediated through sequences in the UTRs of these mRNAs. We tested two variants that had altered 5’UTRs and three that had altered 3’UTRs. In all cases the UTR sequence was shown to regulate luciferase protein levels in a similar way to that predicted by the polysome profiling data. Where the variant had a decreased ribosomal load following polysome profiling, the UTR promoted a decrease in luciferase activity suggesting a role in translational repression. It is likely that the UTRs that are associated with a decreased ribosomal load have cis-acting sequences that confer translational repression. These could take the form of binding sites for miRNA or RNA binding proteins. Alternate 5’UTRs could also contain uORFs which can function to repress translation [38, 39]. The variants that do not have these sequences are likely not targeted for repression. These different UTR sequences have likely evolved to confer additional levels of regulation on specific RNA variants and have the potential to precisely regulate their translation both spatially and temporally. Analysis of the ORF size of the translationally regulated variants suggested that for most variants the differences in ribosomal load were not a consequence of altered ORF size. There were a minority of variants that had significantly longer ORFs that correlated with increased ribosomal load and in these cases the ORF length is likely the cause. For variants that did show a correlation between ORF length and ribosomal load the difference in size was not large enough (less than 25% larger) to result in a shift in the polysome fractions in most cases (Table 1). Interestingly we identified a selection of splice variants that had different translation rates but no change in the UTRs. While some of these candidates will have a decreased ribosomal load due to a decreased ORF length it is likely that the remaining transcripts contain cisacting regulatory sequences within their ORF. miRNAs have been shown to target the ORF of mRNAs [40, 41] so it is possible that the changes in ORF seen in these variants results in altered translational control in addition to altered protein function. Exon 4 and 5 in Ctage5 variant 1 are associated with a decreased ribosomal load compared to variant 3 that skips these exons. While there was no enrichment for rare codons in these exons it is possible that these regions could bind to trans-acting factors or that the sequence itself is inhibiting translation. It will be interesting to determine if these exons can function to regulate translation from within the UTR or if they have to be in the ORF to function. We assessed the ribosomal load of annotated non-coding RNAs and found that a significant number of long non-coding RNAs (lncRNAs) and non-coding RNAs were associated with the ribosomes. The lnc-RNAs include predominantly long intergenic non-coding RNAs (lincRNAs) while the non-coding RNAs include predominantly pseudogenes. The presence of the pseudogenes on the polysomes is in agreement with recent reports that analysed ribosomal footprinting data from a number of different species. [42] Recent studies have demonstrated the presence of many non-coding RNAs on polysomes. Our studies confirm that many noncoding RNAs are in the polysome fraction but we cannot distinguish between RNAs that are actively being translated by the ribosome and those that may be binding the ribosome or other mRNA molecules that are being translated. It is possible that many non-coding RNAs could be associating with the ribosome in a regulatory capacity and so while associated with the ribosome would show no ribosomal footprint. It has been suggested that non-coding RNAs could be being translated to give short peptides but additional studies are needed to elaborate on this idea [32, 42, 43]. Our data confirms the presence of non-coding RNAs on the ribosome and further investigation is needed to determine why they are there. Taken together our data illustrates the importance of addressing the translation rate of individual mRNA variants. We demonstrate that different mRNA variants can have very different translation rates. This confirms and expands on previous reports from human cells and demonstrates variant specific translation rates in ESCs and NPCs. Additional work is needed to determine the mechanisms by which these mRNAs are regulated and their significance for ESC self-renewal and pluripotency. 参考文献:Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation -------------------------------------------------------------- -------------------------------------------------------------- |
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