Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation

Abstract

The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs) and in ESC derived Neural Precursor Cells (NPCs) using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5’UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.

Radiation-induced alternative transcripts as detected in total and polysome-bound mRNA

“…Genes containing more than one splice event per gene (complex) comprised only 13.6% of the genes undergoing alternative splicing. The selective association of alternative transcripts with polysomes suggests that AS influences RNA translation in NSC11 cells, consistent with results obtained from other cell lines [ 15 , 16 ].…”

“…A well-established mechanistic consequence of AS is the formation of transcripts with increased susceptibility to nonsense mediated decay, which ultimately reduces gene expression [ 12 - 14 ]. However, in HEK293T cells and in mouse embryonic stem cells the association of mRNAs with polysomes has been reported to occur in an isoform specific manner [ 15 , 16 ], suggesting a role for AS in the translational control of gene expression.…”

Cytoplasmic long noncoding RNAs are frequently bound to and degraded at ribosomes in human cells

“…We subjected cytoplasmic cellular extracts to polysome profiling, an ultracentrifugation method to identify ribosome-bound RNAs and distinguish transcripts bound to single or multiple ribosomes (Rahim and Vardy 2016). Consistent with previous studies (Zhang et al 2012;Wong et al 2016), extracts were divided into three pools: "heavy polysomal," corresponding to high molecular weight complexes cofractioning with greater than six ribosomes; "light polysomal," cofractioning with two to six ribosomes; and low-molecular weight complexes corresponding to nontranslated, cytoplasmic RNAs (Fig. 1A).…”

Widespread Translational Remodeling during Human Neuronal Differentiation

“…Furthermore, ribosome profiling, which measures the location of ribosomes on mRNAs using RNase-protected footprints, cannot measure translational effects of 3′ UTRs as they are digested. Prior work found that transcript 5′ UTRs mediate isoform-specific translation in mouse ES cells (mESCs) and early NPCs (Wong et al, 2016), but it is unclear if brain-specific 3′ UTR lengthening occurs in six-day-old neural progenitor cells. Therefore, the connection between brain-specific 3′ UTR extension and protein translation, as well as the dynamics of transcript-specific translation during human neuronal differentiation, remain unclear.…”