Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press 1 SYNCRIP, a new player in pri-let-7a processing 2 Ying Chena1, Jingru Chana1, Wei Chena, Jianwei Lia, Meng Suna, Gayathiri Sathyamoorthy 3 Kannana, Yu-Keung Moka, Yuren Adam Yuana,b,†, Chacko Jobichena* 4 5 a Department of Biological Sciences and Centre for Bioimaging Sciences, National 6 University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore 7 b National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, 8 Suzhou Industrial Park, Jiangsu, 215123, China 9 1these authors contribute equally to this work 10 † Passed away on 4th Dec 2018 11 12 * To whom correspondence should be addressed. [email protected] 13 14 15 1 Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press 1 Abstract 2 microRNAs (miRNAs), a class of small and endogenous molecules that control gene 3 expression, are broadly involved in biological processes. Although a number of cofactors that 4 assist or antagonize let-7 miRNA biogenesis are well-established, more auxiliary factors 5 remain to be investigated. Here, we identified SYNCRIP (Synaptotagmin Binding 6 Cytoplasmic RNA Interacting Protein) as a new player for let-7a miRNA. SYNCRIP 7 interacts with pri-let-7a both in vivo and in vitro. Knockdown of SYNCRIP impaired, while 8 overexpression of SYNCRIP promotes the expression of let-7a miRNA. A broad miRNA 9 profiling analysis revealed that silencing of SYNCRIP regulates the expression of a set of 10 mature miRNAs positively or negatively. In addition, SYNCRIP is associated with 11 microprocessor complex and promotes the processing of pri-let-7a. Strikingly, terminal loop 12 of pri-let-7a was shown to be the main contributor for its interaction with SYNCRIP. 13 Functional studies demonstrated that SYNCRIP RRM2-3 domain can promote the processing 14 of pri-let-7a. Structure based alignment of RRM2-3 with other RNA binding proteins 15 identified the residues likely to participate in protein-RNA interaction. Taken together, these 16 findings suggest the promising role that SYNCRIP plays in miRNA regulation, thus 17 providing insights into the function of SYNCRIP in eukaryotic development. 18 19 Key words: SYNCRIP, miRNA biogenesis, pri-let-7a processing 20 Running Title: Role of SYNCRIP in pri-let-7a processing 21 22 23 2 Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press 1 Introduction 2 miRNAs are a class of small, endogenous molecules that post-transcriptionally regulate gene 3 expression according to their sequence complementarity to mRNA, leading to mRNA 4 instability or translational inhibition (Bartel 2009; Fabian et al. 2010). Therefore, miRNAs 5 are broadly involved in various biological processes, and aberrant expression of miRNAs is 6 closely related to a variety of diseases, cancer initiation and progression (Croce 2009; 7 Mendell and Olson 2012; Lin and Gregory 2015). 8 let-7 miRNA, discovered as the first miRNA in human, is a conserved molecule among 9 eukaryotes. A wide range of studies revealed the multiple functions of let-7 miRNA in the 10 regulation of developmental timing and carcinogenesis. In worms, loss-of-function of let-7 11 miRNA affects cell division, blocks the transition from larval to adult stage, causing lethality 12 (Reinhart et al. 2000). In flies, let-7 controls developmental stage transition similar to what 13 was observed in worms (Bashirullah et al. 2003) and the maturation of neuromuscular 14 junctions (Caygill and Johnston 2008; Sokol et al. 2008). In mammalian cells, let-7 regulates 15 timed development, and also acts as a tumor suppressor. 16 Transcribed by RNA polymerase II (RNA pol II) (Lee et al. 2004), canonical primary 17 miRNAs (pri-miRNAs) fold back to form stem-loop structure, which are processed 18 sequentially by Drosha and Dicer in nucleus and cytoplasm, respectively (Hutvagner et al. 19 2001; Ketting 2001; Lee et al. 2003). In the nucleus, Drosha-mediated cleavage generates 20 ~65nt, hairpin-structured precursor miRNAs (pre-miRNAs) which are later transported into 21 cytoplasm by Exportin 5 in a Ran-GTP-dependent manner (Yi et al. 2003; Bohnsack 2004; 22 Lund 2004). In the cytoplasm, pre-miRNAs are further cropped into miRNA duplex, and one 23 strand of which, the mature miRNA, is incorporated into RISC (RNA-induced silencing 24 complex). The mature miRNA serves as a guide for Agonaute2 (AGO2) to drive the 25 molecular machinery for RNAi (Kobayashi and Tomari 2016). miRNA biogenesis is an 3 Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press 1 intricate, multi-step process where regulation could occur to fine tune the expression of 2 miRNA in accordance with differentiation and development requirements. Up to date, a 3 series of sequence determinants on miRNA precursors are known and a number of protein 4 regulators have been well-recognized that define microprocessor targets and fine tune 5 miRNA expression level, respectively (Gebert and MacRae 2019; Michlewski and Caceres 6 2019). Especially the terminal loop, a unique but conserved feature for miRNA precursors, 7 not only contains determinants to orientate microprocessor but provides a major landing 8 platform attracting RNA binding proteins (RBPs) to exert regulatory function. Of interest, it 9 was reported earlier that only RRMs are sufficient to induce conformational destabilization of 10 stem-loop structure, which promotes or inhibits nuclear processing (Chen et al. 2016; 11 Kooshapur et al. 2018). In addition to the terminal loop, several regulators recognize specific 12 sequences embedded in flanking region, for example, QKI5 promotes pri-miR-124 13 processing by recognizing a distal element away from the stem-loop structure (Wang et al. 14 2017). For let-7 miRNA, a number of modulators have been identified, such as Lin28, 15 hnRNP A1 and KSRP, which dock on to its terminal loop and regulate its expression 16 positively or negatively. However, our understanding of post-transcriptional regulation in 17 miRNA biogenesis remains at an early stage and more auxiliary factors await investigation 18 regarding the incredible functions of miRNAs in cellular pathways. 19 SYNCRIP (also known as hnRNP Q or NSAP1) is an evolutionally conserved RNA binding 20 protein across eukaryotic organisms and participates in several cellular pathways and diseases, 21 especially in neuronal and muscular development. On the RNA side, SYNCRIP has multiple 22 roles in the control of RNA metabolism through recognizing a variety of sequences and 23 regulating pre-mRNA splicing, translation, transport as well as degradation. For example, 24 SYNCRIP modulates alternative splicing of SMN2 transcript, which may compensate the loss 25 of SMN1 in spinal muscular atrophy patients (Chen et al. 2008). In addition, SYNCRIP 4 Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press 1 specifically recognizes AUAAUC sequence to regulate the localization of a set of mRNAs, 2 which may further modulate neuronal morphogenesis (Chen et al. 2012). Of particular 3 interest, recently, SYNCRIP was revealed to trigger a vehicle machinery which partitions 4 miRNAs out of cytoplasm to exosome. The molecular principles include wide recognition of 5 mature miRNAs containing hEXO sequence (GGCU/A) and mediation of miRNAs 6 enrichment in exosomes (Santangelo et al. 2016; Hobor et al. 2018). 7 Although SYNCRIP has been implicated in various aspects of pre-mRNA splicing, RNA 8 localization as well as mRNA translation, its effect on pri-miRNA processing has not been 9 established. Here, we identified that pri-let-7a interacts with SYNCRIP both in-vivo and in- 10 vitro and this interaction contributes to increased expression of let-7a miRNA. In addition, 11 miRNA microarray results revealed that the regulation controlled by SYNCRIP is not limited 12 to let-7a miRNA; in fact, a subset of miRNAs are regulated by SYNCRIP. The molecular 13 basis underpinning this regulation includes the interaction with DiGeorge Syndrome Critical 14 Region gene 8 (DGCR8) and enhanced microprocessor-mediated processing. Of particular 15 interest, we found that SYNCRIP binds to the terminal loop of let-7a precursors and we 16 identified one of its recognized sequences, UAGAAU, on the apical loop of let-7a precursors. 17 Furthermore, studies on domain function showed that the two tandem RNA recognition 18 motifs (RRMs; RRM2-3) of SYNCRIP harbor the function of the full-length protein to 19 promote pri-let-7a processing. Notably, SYNCRIP is a new regulator in let-7a miRNA 20 biogenesis, which sheds light on SYNCRIP-mediated developmental abnormality. 21 Materials and Methods 22 Cell culture 23 Human HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) 24 supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 unit/ml penicillin, 100 25 ug/ml streptomycin (Gibco). Cells were maintained at 37°C in the presence of 5% CO2. 5 Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press 1 2 Transfection 3 Human HEK293 cells were transfected with plasmids using polyethylenimine (PEI, 4 Polysciences) as a transfection reagent. Once the cells grew to 80-90% confluency, 5 transfection was performed with the ratio of plasmid to PEI as 1:2. After 48 hours post- 6 transfection, the cells were washed with phosphate buffered saline (PBS) before proceeding 7 to the downstream experiments. 8 9 Construction of vectors used in this study 10 To achieve efficient knockdown of SYNCRIP, we used pSuper vector to express shRNA. To 11 overexpress SYNCRIP in HEK293 cells, we used pXJ40-myc or pXJ40-flag vector which 12 expresses fusion protein with myc or flag tag at the N-terminus. To overexpress pri-miRNA 13 in living cells, we used modified pcDNA5/tRSA.