Potential Functions Of Eukaryotic SRPK1 In MiRNA Biogenesis By Regulating The Phosphorylation Of Drosha

miRNAs are a class of short single-stranded non-coding RNAswith a length of20-24 nt that are widely spread in animals and plants.They play key roles in gene regulation,and involve in almost all known physiological and pathological processes,thus it is important to dissect the regulation mechnisms of their biogenesis.Drosha,a nuclear RNase III that form a microprocessor with DGCR8 in the nucleus and librates the pre-mi RNAs in mi RNA biogenesis,contains a RS-rich domain in its N-terminus that might be a substrate of SR protein-specific kinase SRPK1.SRPK family is a special Ser/Thr kinase that includes two members in animals: SRPK1 and SRPK2.They can phosphorylate the RS-rich domain of SR proteins in a precessing way in cytoplasm thus play an important role in m RNA splicing.It has been demonstrated that SRPK1 can be immunoprecipitated with Drosha,indicating their possible present in the same complex.Our prelimitary results also showed that SRPK1 could interact with Drosha in vivo.Furthermore,results from large-scale mass spectrometry from database Phospho Site Plus revealed the multiple phosphorylation sites in its N-terminal RS-rich domain of Drosha.Combined all these results,we proposed that SRPK1 may act as a potential kinase and phosphorylate Drosha directly in mice.This dissertation is to confirm the phosphorylation of Drosha by SRPK1,define the possible phosphorylationsites,prove their importance in mi RNA biogenesis and dissect the underlying mechanisms of how the SRPK1-induced phosphorylation changes of Drosha affect its functions in mi RNA processing.These will help us in understanding the biogenesis and regulation of mi RNAs.To achieve this,we first confirmed whether SRPK1 could directly phosphorylate Drosha by in vitro kinase assay.We purified the His-tagged Drosha and SRPK1 protein that induced independently in E.coli and conducted the in vitro kinase assay.The results showed that the SRPK1 could phosphorylate Drosha in vitro.After confirming the phosphorylation of Drosha by SRPK1,we then carried out a mass spectrometry analyses on Drosha followed the in vitro kinase assay.We identified 4 phosphorylation sites: S201,S237,S300 and S302,which are all present in RS-rich domain of Drosha.We made S to A mutations in all four sites,respectively and again overexpressed and purified them in E.coli.The following in vitro kinase assay showed that the phosphorylation levels of Drosha were significantly reduced after those mutations(data not shown in this dissertation).From all these results,we confirmed that SRPK1 could act as a kinse to directly phosphorylate Drosha in vitro.Next,we started to verify whether SRPK1 could actually affect the mi RNA biogenesis.To achieve this,we conducted a genome-wide mi RNA profiling assay.We constructed a mi RNA library from the total RNAs extracted from WT and SRPK1 knockout MEF cells,respectively.After the high throughput sequencing,the results were plotted to the mi RBASE database and analyzed.It showed that in the 239 mi RNAs indentifed in the sequencing,the expression of 36 mi RNAs was changed after SRPK1 KO: 13 of them were increased while 23 of them were decreased.The following Q-PCR on selected mi RNAs confirmed the sequencing results.Combined all rhese results,we demonstrated that SRPK1 did affect the mi RNA level in cells,that is to say,SRPK1 involve in the mi RNA biogenesis,and this may function through Drosha.Finally,we further studied the possiblemechanismsunderlyinghow SRPK1 affect the mi RNA biogenesis or how SRPK1 modify the Drosha functions through phosphorylation.It has beed demonstrated that phosphorylation level of SR proteins can regulatetheir cellular localization: dephosphrylated SR proteins will retain in the cytoplasm thus fail to function in m RNA splicing.Because Drosha contains a RS-rich domain in its N-terminus and may consider as a special SR protein,we first verified whether the phosphorylation changes could affect the cellular localization of Drosha.We transfected GFP-tagged WT or different kinase-dead(S to A)mutants in 293 T cells after knockdown of SRPK1 by RNAi and followed by immunostaining to check the localization of Drosha.The experiments proved that when SRPK1 was knocked down or phosphorylation sites of Drosha were mutanted,the subcellular localization of Drosha in 293 T was altered: they leaked out from nucleus to cytoplasm..In addition,it has been demonstrated that RS-rich domain in SR proteins isrelated to its protein interactions.Therefore,we also checked whether the phosphorylation changes of Droshacould affect its interaction with DGCR8 and therefore affect its function in mi RNA biogenesis.To test this,we first expressed GFP-tagged WT or kinase-dead mutants of Droshain 293 T cells after SRPK1 knockdown.The following co-IPexperiments demonstrated that the binding between Drosha's triple mutant(S221A/S300A/S302A)and DGCR8 was significantly reduced.In general,the results from this dissertationconfirmed that SRPK1 could directly phosphorylate Drosha in mice,identified 4 phosphorylation sites in its SR-rich domain,proved their importance in mi RNA biogenesis.Furthermore,we explored the possible mechanisms underlying the effects of SRPK1 on mi RNA processing,demonstratedthat SRPK1 could alter the subcellular localization of Drosha and its interaction with DGCR8 by changing the phosphorylation level of Drosha.SRPK1 is one of the key players in m RNA splicing and cell signal transduction,finding its potential functions in regulating mi RNA biogenesis will help us strengthen our research in this field and find the possible effective treatment for mi RNA misfunctional diseases.