Regulation Of Drosha By SRPK1 In Mouse Cells And Its Possible Mechanisms

mi RNA is a class of single-stranded small non-coding RNAs,which are around20?24 nt long.They are important regulators in gene expression and involve in almost all known physiological and pathological processes including cancer.To study and understand the mechanisms of mi RNA synthesis and regulation are of great practical significance for dissecting the underlying pathogenic mechanisms of diseases and developing effective drugs and treatment methods accordingly.Drosha is a nuclear-localized RNase III,participates in the hydrolytic process of pri-mi RNAs to pre-mi RNAs,and is one of the most important enzymes in mi RNA processing.Sequence analysis showed that Drosha contains a RS-rich region at its N-terminus,which is similar to the RS domain in SR proteins,an important group of splicing regulators.Studies have shown that phosphorylation levels of the RS domain in SR proteins are closely related to their functions,and only hyper-phosphorylated SR proteins can be shuttled back to the nucleus to perform their functions.SR protein-specific kinase(SRPKs)is a special class of serine/threonine kinases.Both famlily members in animals,SRPK1 and SRPK2,can progressively phosphorylate SR proteins in the cytoplasm and therefore are crucial for maintaining the functions of SR proteins.We propose that Drosha might be phosphorylated by SRPK1,as a “special” SR protein,based on the following facts.First,it has been demonstrated that SRPK1 can be immunoprecipitated with Drosha,and is the only kinase in the immunoprecipitants.Second,our preliminary data showed that SRPK1 could interact with Drosha.Third,mass spectrometry data obtained from Phospho Site Plus Database revealed multiple phosphorylation sites are present in the RS-rich domain of Drosha.This dissertation aims to confirm whether Drosha is a potential substrate of SRPK1,to analyze how the phosphorylation level of Drosha affects mi RNA synthesis,and to further explore the possible molecular mechanisms underlying.IVFirstly,we confirmed that SRPK1 interact with Drosha and directly phosphorylate it in vitro by pull-down and in vitro kinase assays.Subsequent mass spectrometry analysis identified four phosphorylation sites in its RS-rich domain:S201,S237,S300 and S302.The kinase-dead mutant(S to A)designed accordingly could no longer be phosphorylated by SRPK1 in vitro.These results demonstrated that SRPK1 can act as a kinase and phosphorylate the RS-rich domain of Drosha directly and the four phosphorylation sites,S201,S237,S300 and S302,are main phosphorylation sites of SRPK1.Secondly,we conducted a genome-wide mi RNA profiling assay to determine whether SRPK1 could affect mi RNA biogenesis.The results showed that the expression levels of 36 out of 239 mi RNAs we detected changed significantly after SRPK1 knockout: 23 of them decreased wherase 13 of them increased.The subsequent Q-PCR confirmed the reliability of the mi RNA profiling.Taken all together,we demonstrated that SRPK1 knockout could affect the biosynthesis of some mi RNAs.The following Q-PCRs using the mi RNAs with significant changes after SRPK1 KO confirmed that the changes occurred during the process that pri-mi RNAs are hydrolyzed to pre-mi RNAs,which is the reaction where Drosha functions in.Lastly,we explored the possible mechanisms of how SRPK1 affects mi RNA biogenesis using immunofluorescence and immunoprecipitations.We found that when SRPK1 is knocked down or phosphorylation sites of Drosha were mutated,the subcellular localization of Drosha changed,they diffused from nucleus into cytoplasm.Meanwhile,the interaction between Drosha and DGCR8 was decreased,indicating the interaction between these two depends on the phosphorylation level within the RS-rich domain of Drosha.In another word,SRPK1-induced phosphorylation of Drosha could affect its subcellular localization and interaction with DGCR8,thus regulates their functions in mi RNA biofgenesis.The results of this dissertation demonstrate that,1,SRPK1 can function as a Drosha kinase and phosphorylate it directly in vitro,4 phosphorylation sites were identified in Drosha by mass spectrometry and they all,as expected,locate within the RS-rich domain;2,SRPK1 KO can affect the hydrolysis of pri-mi RNAs to pre-mi RNAs,which is where Drosha functions in mi RNA biogenesis;3,SRPK1 can affect the interactions between Drosha and DGCR8,regulate the subcellular localization of Drosha by altering its phosphorylation level,diffusing Drosha from nucleus to cytoplasm.These results fully confirm that SRPK1 affect mi RNA biosynthesis by phosphorylating Drosha.SRPK1 is an important regulator in m RNA splicing and cell signal transduction,finding its possible regulating mechanisms in mi RNA biogenesis will facilitate the research on gene expression regulation and has important theoretical and clinical significance in dissecting the pathogenic mechanisms of mi RNA-related disorders.