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The RNA Binding Protein EWS Is Broadly Involved in the Regulation of Pri-Mirna Processing in Mammalian Cells

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The RNA Binding Protein EWS Is Broadly Involved in the Regulation of Pri-Mirna Processing in Mammalian Cells UC San Diego UC San Diego Previously Published Works Title The RNA binding protein EWS is broadly involved in the regulation of pri-miRNA processing in mammalian cells. Permalink https://escholarship.org/uc/item/1md634kt Journal Nucleic acids research, 45(21) ISSN 0305-1048 Authors Ouyang, Huiwu Zhang, Kai Fox-Walsh, Kristi et al. Publication Date 2017-12-01 DOI 10.1093/nar/gkx912 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Published online 9 October 2017 Nucleic Acids Research, 2017, Vol. 45, No. 21 12481–12495 doi: 10.1093/nar/gkx912 The RNA binding protein EWS is broadly involved in the regulation of pri-miRNA processing in mammalian cells Huiwu Ouyang1, Kai Zhang1, Kristi Fox-Walsh2, Yang Yang1, Chen Zhang1, Jie Huang1, Hairi Li2, Yu Zhou1,3,* and Xiang-Dong Fu1,2,* 1State Key Laboratory of Virology and Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, China, 2Department of Cellular and Molecular Medicine, Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093-0651, USA and 3Institue of Advanced Studies, Wuhan University, Wuhan 430072, China Received December 12, 2016; Revised September 07, 2017; Editorial Decision September 26, 2017; Accepted September 27, 2017 ABSTRACT and RNA processing (7) and mediating splice site selection during pre-mRNA splicing (8–11). Consequently, knockout The Ewing Sarcoma protein (EWS) is a multifaceted of these RBPs causes severe developmental abnormality in RNA binding protein (RBP) with established roles in mice (12,13). Importantly, various chromosome transloca- transcription, pre-mRNA processing and DNA dam- tion events that involve EWS and mutations in both EWS age response. By generating high quality EWS–RNA and FUS/TLS have been linked to specific human diseases interactome, we uncovered its specific and preva- (14,15). lent interaction with a large subset of primary mi- Given the ability of individual TET family members to croRNAs (pri-miRNAs) in mammalian cells. Knock- bind RNAs, multiple groups have performed crosslinking down of EWS reduced, whereas overexpression en- immunoprecipitation coupled with deep sequencing (CLIP- hanced, the expression of its target miRNAs. Bio- seq) to characterize their RNA binding profiles on both chemical analysis revealed that multiple elements in cellular and animal models (16,17). The initial analysis by PAR-CLIP on HEK293 cells showed related, but distinct target pri-miRNAs, including the sequences flank- RNA binding profiles of FUS/TLS, EWS and TAF15 (18). ing the stem–loop region, contributed to high affinity This study also revealed a general association of these RBPs EWS binding and sequence swap experiments be- with 3 splice sites in pre-mRNAs and a preference for both tween target and non-target demonstrated that the G-rich and AU-rich sequences. However, the association of flanking sequences provided the specificity for en- these RBPs with 3 splice sites was not seen by a separate hanced pri-miRNA processing by the Microproces- CLIP study of EWS on HeLa cells, which instead showed sor Drosha/DGCR8. Interestingly, while repressing enriched RNA binding near EWS-regulated 5 splice sites Drosha expression, as reported earlier, we found that (10). Two independent genome-wide analyses of FUS/TLS EWS was able to enhance the recruitment of Drosha in mouse and human brain also found its prevalent coat- to chromatin. Together, these findings suggest that ing on long pre-mRNA transcripts; however, most binding EWS may positively and negatively regulate miRNA events detected in these studies did not seem to occur near induced alternative splicing events in FUS/TLS deficient biogenesis via distinct mechanisms, thus providing cells (8,11). While it has been unclear about the sources of a new foundation to understand the function of EWS such discrepancies, the seemly degenerative sequence pref- in development and disease. erence for the TET family members might be explained by the observation that FUS/TLS appears to bind certain sec- INTRODUCTION ondary structures in RNAs, rather than specific motifs in exposed single-stranded RNA regions (18). More impor- EWS belongs to the TET family of RNA binding pro- tantly, the biological meaning of most detected RNA bind- teins (RBPs), consisting of FUS/TLS, EWS, and TAF15 ing events has been poorly understood. (1,2). These RBPs have been implicated in multiple lay- We were initially motivated to investigate various in- ers of regulated gene expression via their roles in modu- consistencies among published genome-wide RNA inter- lating transcription (3–6), coupling between transcription *To whom correspondence should be addressed. Tel: +1 858 534 4937; Email: [email protected] Correspondence may also be addressed to Yu Zhou. Tel: +86 27 68756749; Email: [email protected] C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 12482 Nucleic Acids Research, 2017, Vol. 45, No. 21 actomes by the TET family members. Instead of relying contains a 3 degenerate anchor and a universal tag se- on mining the existing datasets, we generated our own quence at the 5 end, allowing quantitative analysis of ma- high quality EWS CLIP-seq libraries on HeLa cells and ture miRNA by real-time PCR using the universal primer noted prevalent interaction of EWS with a large number and a miRNA-specific primer. Quantitative PCR was car- of expressed pri-miRNAs, reminiscent of FUS/TLS bind- ried out with 1:100 dilution cDNA, 2× SYBR Green PCR ing to hairpin-containing RNAs as reported earlier (18). Mix, 10× miScript universal primers included in the kit, in We therefore decided to focus on this new lead in the cur- combination with 10× miRNA specific primers (listed in rent study because it has been reported that a large num- Supplementary Table S1). The U6 snRNA primer from Qi- ber of miRNAs were induced while others suppressed in agen was used for normalization and Ct was calculated to EWS knockout mouse embryonic fibroblasts (MEFs)19 ( ). derive relative expression. Interestingly, EWS deficiency has also been linked to ele- vated Drosha expression at both the mRNA and protein levels, and because Drosha is the catalytic subunit of the Microprocesssor, which is recruited to chromatin to facil- Plasmid construction, luciferase assay, protein purification itate co-transcriptional pri-miRNA processing in the nu- cleus (20,21), increased Drosha may therefore account for Myc-tagged EWS cDNA at the N-terminus was generated the induction of a specific set of miRNAs (19). However, by PCR using specific primers (listed in Supplementary Ta- how EWS deficiency would also cause the repression of ble S2) and inserted into pcDNA3.0 between EcoR I and other miRNAs has remained unknown. Xho I sites for EWS overexpression in transfected HeLa We now provide evidence for a direct role of EWS in cells. enhancing pri-miRNA processing by the Microprocessor, For MS2-based capture experiments, the MS2 stem–loop thus joining EWS to the growing list of RBPs involved in sequence was excised from the plasmid 5U3M described modulating miRNA biogenesis in mammals (22–24). Un- previously (30) by restriction digestion with Hind III and like other RBPs involved in modulating miRNA biogene- Xho I, ligated to PCR-amplified pri-miRNA containing the sis described earlier, EWS appears to bind and modulate Xho I and Not I sites, and then inserted the product into processing of a large number of pri-miRNAs. Coupled with pcDNA3.0 at Hind III and Not I sites. The plasmid for ex- EWS-mediated Drosha repression, this RBP appears to be pressing the EGFP-MS2 fusion protein was as described capable of both stimulating and inhibiting miRNA biogen- previously (30). The primers used in MS2-Pri-miRNA plas- esis, but via distinct mechanisms, which we have dissected mid construction are listed in Supplementary Table S2. in this study. The newly elucidated function of EWS adds a To construct the Renilla luciferase reporters for mea- new dimension in understanding the mechanisms underly- suring pri-miRNA processing in transfected cells, individ- ing EWS mutation-induced cancers (5,25,26) and neurode- ual pri-miRNA sequences were first PCR amplified by us- generative diseases (27). ing specific primers (listed in Supplementary Table S2) and cloned into psiCHECK™-2 Renilla 3UTR between the Xho I and Not I sites. MATERIALS AND METHODS Pri-miRNAs used for gel shift and in vitro processing as- says were all transcribed from pcDNA3.0 clones contain- Cell culture, transfection, antibodies, RT-qPCR of miRNAs ing individual pri-miRNAs generated by PCR using spe- HeLa cells were grown in Dulbecco’s modified Eagle’s cific primers (listed in Supplementary Table S2). To con- medium (DMEM) supplemented with 10% newborn struct various chimeric pri-miRNA plasmids, individual ◦ bovine serum (Gibco) at 37 Cin5%CO2. RNAimax fragments were PCR amplified from the pcDNA3.0-pri- and Lipo2000 (Life Technology) were used for siRNA miR-222 or pcDNA3.0-pri-miR-23a plasmid, ligated, and and plasmid transfection, respectively, according amplified in transformed bacteria. to manufacturer’s instructions. The siRNA against For luciferase assays, HeLa cells were seeded in 24-well Drosha (5-AACGAGUAGGCUUCGUGACUU- plates and co-transfected with 25 ng of luciferase reporter 3) was prepared based on published sequences and/or 300 ng pcDNA3.0-based expression vector. After (28), and two independent siRNAs against EWS 48 h, cells were harvested for luciferase assays using the (5-AUGAUCUGGCAGACUUCUUUA-3;5- Luciferase Assay System (Promega, E1500).
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