Final Report Summary - MIRNA BIOGENESIS (Auxiliary factors involved in the post-transcriptional regulation of microRNA expression.)
a) Post-transcriptional regulation of mammalian LINE-1 retrotransposons by the microprocessor complex.
The Drosha-DGCR8 complex (microprocessor) has a well-characterised role in microRNA (miRNA) biogenesis (1, reviewed in 2, 3). DGCR8 contains two double-stranded RNA binding motifs that recognise the RNA substrate, whereas Drosha functions as the endonuclease. Thus, the microprocessor complex cleaves hairpin structures embedded in primary transcripts in the nucleus (pri-miRNAs) that are further processed by Dicer in the cytoplasm to generate the mature miRNAs.
The aim of this study was to identify RNA targets of the microprocessor complex. Using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) of DGCR8 in human 293 cells, we have shown that a high proportion of the identified targets correspond to human Transposable Elements. Among them, targets derived from human LINE-1 elements constituted ~ 35 % of the reads. Upon analysis, we observed that CLIP reads distributed along the consensus sequence of a RC-L1, with main peaks located at the 5'UTR, which is predicted to form a stable secondary structure.
Remarkably, in vitro and in vivo analyses revealed that the microprocessor complex process primary transcripts resembling the 5'UTR of RC-L1s. In addition, we found that DGCR8/Drosha controlled the abundance of L1mRNA and the LINE-1 encoded ORF1 protein in human and mouse cells. Using a cell cultured based L1 retrotransposition assay, we also observed that the microprocessor complex negatively regulates L1 retrotransposition. Interestingly, this effect was partially abolished when the 5'UTR was removed from the engineered human L1.
In sum, these results suggest that the microprocessor complex act to regulate L1 retrotransposition at a post-transcriptional level, as a defender of human genome integrity against endogenous retrotransposons. The results from this project form part of a Research Article currently in preparation.
On the other hand, a frequent human disease known as DiGeorge (DG) Sindrome or 22q11. 2 deletion is characterised by the deletion of a fragment of chromosome 22 during meiosis that includes the DGCR8 gene (4). DG affects 1/4000 newborns, but it has different penetrance depending on the number of genes deleted at 22q11. 2. However, DG patients suffer from cardiac malformations, schisophrenia, and immunological problems among others (reviewed in 4-6). It stands to reason that most of the symptoms are related to lack of key miRNAs during human embryonic development. However, is also likely that some of the symptoms observed in these patients might be related to exacerbated L1 retrotransposition. The results found during this project have opened a new research topic in the field. The researcher plan to study the impact of LINE-1 retrotranposition in early human development in patients affected with human disease DiGeorge Sindrome.
b). The SR protein SRSF1 couples splicing and translation.
The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. The SR protein prototype, SRSF1 (also known as SF2/ASF or SFRS1), has been initially characterised as a splicing factor but also shown to mediate post-splicing activities such as mRNA export and translation (reviewed in 7). We had previously shown that SRSF1 promotes translation initiation of bound mRNAs by suppressing the activity of 4E-BP, a competitive inhibitor of cap-dependent translation. This activity is mediated by interactions of SRSF1 with components of the mTOR signalling pathway. These findings suggested the model whereby SRSF1 functions as an adaptor protein to recruit the signalling molecules responsible for regulation of cap-dependent translation of specific mRNAs (8). In order to dissect the importance of SRSF1 in translational control, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. A group of more than one thousand mRNAs shifts from the subpolysomal fraction to the heavier polysomal fractions upon SRSF1 overexpression. Interestingly, one third of these translationally regulated mRNAs were previously identified as bona fide RNA targets of this SR protein by CLIP-seq (9). Bioinformatics analyses showed that these mRNAs encode for proteins involved in cell cycle regulation and transcription, which could partially explain the proposed role of SRSF1 as an oncogene. Moreover, higher expression of those proteins upon SRSF1 overexpression has been confirmed using SILAC.
Finally, we analyzed, using exon juntion expression arrays, changes in alternative splicing in response to different levels of SRSF1 protein. Interestingly, a significant proportion of those mRNAs that display alternative splicing changes upon SRSF1 overexpression are also translationally regulated, suggesting that SRSF1 influences several steps of an mRNA life. In summary, these data provide insights on the complex role of SRSF1 in the control of gene expression and its implications in cancer. The results from this project form part of a Research Article in preparation.
1-Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes & Development 18, 3016-3027 (2004).
2-Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233 (2009).
3-Heo, I. & Kim, V. N. Regulating the regulators: posttranslational modifications of RNA silencing factors. Cell 139, 28-31 (2009).
4-Demczuk, S. et al. Excess of deletions of maternal origin in the DiGeorge/velo-cardio-facial syndromes. A study of 22 new patients and review of the literature. Hum Genet. 96, 9-13 (1995).
5-Qurashi, A. & Jin, P. Small RNA-mediated gene regulation in neurodevelopmental disorders. Curr Psychiatry Rep. 12, 154-161 (2010).
6-Walter, E., Mazaika, P. K. & Reiss, A. L. Insights into brain development from neurogenetic syndromes evidence from fragile X syndrome, Williams syndrome, Turner syndrome and velocardiofacial syndrome. Neuroscience 164, 257-271 (2009).
7-Long, JC., Caceres, JF. The SR protein family of splicing factors: master regulators of gene expression. Biochem J. 417 (1): 15-27 (2009).
8-Michlewski G, Sanford JR, C?ceres. JF. The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1. Mol Cell. 30 (2): 179-89 (2008).
9-Sanford et al. Splicing factor SFRS1 recognises a functionally diverse landscape of RNA transcripts. Genome Research. 19, 381-394 (2009)