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Genome-wide Studies reveal that LIN28 Enhances the Translation of Genes Important for Growth and Survival of Human Embryonic Stem Cells

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From the March 2011 Issue of Stem Cells

Paper Commentary by Stuart P. Atkinson

The RNA binding protein LIN28 (or LIN28A) is highly expressed in human embryonic stem cells (hESCs) and is often used in the generation of induced pluripotent stem cells (iPSCs) (Yu et al.). It is known to play a role in inhibiting the maturation and promoting the degradation of the Let7 family of microRNAs which are known to promote the expression of genes involved in differentiation. However multiple Let7 independent roles for LIN28 have also been observed, and have prompted a study from the laboratory of Yingqun Huang at the Yale Stem Cell Center presented in the March edition of Stem Cells.

Peng et al. began by studying the effect of LIN28 knockdown in hESC by siRNA and observed a decrease in the number of viable cells and increased apoptosis. These data, alongside previous work established in mouse (Xu et al.) suggest that LIN28 is important for the growth and survival of hESCs. To study how LIN28 may mediate these effects, target RNAs were sought after using an RNA-immunoprecipitation (RNA-IP) assay using a LIN28 specific antibody, subsequent cDNA synthesis and deep sequencing using the Illumina platform. RNAs showing over a 2.5-fold enrichment in the LIN28 IP compared to a control IP were analysed (268 genes) and gene ontology analysis showed that genes encoding RNP proteins (including splicing factors), genes involved in translation (including ribosomal proteins and translation initiation and elongation factors) and genes involved in cellular metabolism were over-represented. Meanwhile, genes involved in membrane receptor activity, DNA-binding and transcription were under-represented. These results are consistent with the role of LIN28 in regulating cellular growth and metabolism in a positive manner. This was further validated by the study of selected LIN28 targets (OCT4 (POU5F1), HMGA1, EEF1G and RPS13) which showed that when LIN28 levels were reduced by siRNA (to 15%), protein levels of targets fell by 40-70% in H1 and PA-1 hESCs, although RNA levels remained relatively unaltered.

This suggests that LIN28 may be stimulating the translation of these target mRNAs. If this is so, polysomes (clusters of ribosomes bound to an mRNA molecule) containing LIN28 should be also enriched in target mRNA molecules. Further analysis showed this to be true, with OCT4, EEF1G, HMGA1 and RPS13 mRNAs enriched by at least 2-fold while b-Actin (ACTB), a control mRNA, was not. Reduced association of the target mRNAs with polysomes upon LIN28 down-regulation was also observed. The question of how this actually occurs now arises. Previous studies by the same group (Qiu et al.) described a LIN28-responsive element (LRE) in the OCT4 coding sequence which allows for LIN28 mediated stimulation of translation, analysed via a luciferase reporter assay. LREs were then subsequently discovered in EEF1G, HMGA1 and RPS13 coding sequences suggesting that LIN28 does allow for the increased translation of these mRNAs into protein by a direct mechanism. The LREs described here were at least 95 nucleotides in length suggesting that LIN28 may recognise a specific 3-dimensional RNA structure rather than a specific sequence.

Overall, these results show a direct role for LIN28 in the increase in translation of mRNAs known to mediate the growth and survival of hESCs. The authors do however note the existence of a number of interesting interactions that were not fully studied in their current work and may be of further interest. These include a potential for LIN28 in chromatin regulation through HMGA1 and CHMP2A and in pre-mRNA splicing or RNA metabolism through genes such as PUF60, MAGOH, RAN and the arginine methyltransferases PRMT1 and PRMT2. It seems that further roles for LIN28 are likely to be uncovered soon.

 

References

Induced pluripotent stem cell lines derived from human somatic cells.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA.
Science. 2007 Dec 21;318(5858):1917-20.

Genome-wide Studies reveal that Lin28 Enhances the Translation of Genes Important for Growth and Survival of Human Embryonic Stem Cells.
Peng S, Chen LL, Lei XX, Yang L, Lin H, Carmichael GG, Huang Y.
Stem Cells. 2011

Lin28 modulates cell growth and associates with a subset of cell cycle regulator mRNAs in mouse embryonic stem cells.
Xu B, Zhang K, Huang Y.
RNA. 2009 Mar; 15(3):357-61.

Lin28-mediated post-transcriptional regulation of Oct4 expression in human embryonic stem cells.
Qiu C, Ma Y, Wang J, Peng S, Huang Y.
Nucleic Acids Res. 2010 Mar; 38(4):1240-8.