Mechanism Of Small RNA Methyltransferase CrHEN1 In MiRNA Biosynthesis In Chlamydomonas

micro RNAs(mi RNAs)are a class of endogenous about 21–24 nucleotides(nt)non-coding small RNAs(s RNAs)that generally promote degradation and/or translation inhibition of target m RNAs with post-transcribe regulation.HEN1 protein was the first discovered in Arabidopsis thaliana with methyltransferase activity,methylating double-stranded mi RNA,so that,it promotes mi RNA stability,and prevent mi RNA from being degraded by urylation at the 3 'end.HEN1 is a kind of more conservative protein.Its homologous protein exists in plants,animals and some bacteria,but their biological function is slightly different.Until 2007,a large number of mi RNA was first found in Chlamydomonas reinhardtii,but the mechanism of methylation modification in the mi RNA biosynthetic pathway has not been reported yet.Therefore,in this study,first,we compared the sequencing results of Chlamydomonas reinhardtii Crhen1 mutants with wild type and found that most mi RNAs in Crhen1 mutants were down-regulated.After verification by q RT-PCR and Northern blot,it was found that mi RNA expression was consistent with the sequencing results,confirming that CrHEN1 plays an important role in the mi RNA biosynthesis pathway in Chlamydomonas reinhardtii.1.By analyzing the omics sequencing data,the mi RNAs with down-regulated expression of Crhen1 mutant were screened,and mi RNA probes were designed in Chlamydomonas reinhardtii.After the ?-elimination of the mi RNA of Crhen1 and wild type,Northern blot gel electrophoresis was performed.The results showed that CrHEN1 has methyltransferase activity,and there was a methylation modification process in the biosynthesis process of Chlamydomonas reinhardtii mi RNA.2.By constructing the GFP-tagged pj1CF8-CrHEN1 vector,the recombinant vector was transformed into Chlamydomonas reinhardtii CC849 by bead milling method to obtain Chlamydomonas transformants.The observation of Chlamydomonastransformants under a laser confocal microscope revealed that most of the fluorescence was located in the nucleus of Chlamydomonas reinhardtii and a small part was in the cytoplasm.These observations indicates that the mi RNA methylation modification occurs in the nucleus in Chlamydomonas reinhardtii.3.The CrHEN1 domain(Hd)and DCL3 domain(dicer)proteins were obtained through the E.coli prokaryotic expression system,and the Hd and dicer were purified and concentrated.ITC technology was used to verify that the interaction between Hd and dicer occurred.This indicates that CrHEN1 and DCL3 work synergistically in the process of mi RNA biosynthesis in Chlamydomonas reinhardtii.4.Through the heterologous recovery experiment of CrHEN1 in Arabidopsis hen1 mutant,CrHEN1 can greatly restore the phenotype of Arabidopsis hen1 mutant,further illustrating the biological function of methylation modification of CrHEN1 in Chlamydomonas reinhardtii.In summary,the results of this study shows that CrHEN1 is methylated in the nucleus of the mi RNA biosynthetic pathway,and CrHEN1 and DCL3 work synergistically in Chlamydomonas reinhardtii.This experiment preliminarily explained the regulation mechanism of CrHEN1 in mi RNA biosynthesis,which laid a theoretical foundation for further elucidating the regulation of small RNA methylation in unicellular organisms,and provided an important theoretical basis for the follow-up research on the mechanism of mi RNA biosynthesis in Chlamydomonas reinhardtii.