PRMT5 Regulates Alternative Splicing And Function Of Photorespiration Related Genes

PRMT5 encodes a type II protein arginine methyltransferase,which is involved in regulating the cleavage of different gene pre-mRNAs and plays an important role in plant to response to biotic and abiotic stresses.To further understand the function of alternative splicing regulated by PRMT5,we used Col-0 and prmt5-2 as materials and used RNA-seq technology to analyze that the PRMT5 led lots of genes to create aberrant alternative splicing events and we focused on the analysis of photorespiration related genes.The aberrant alternative splicing of photorespiration genes,exploring the possible function of aberrant alternative splicing,provides a new tool on the study of the mechanism between the alternative splicing and photorespiration pathways.The main results obtained are as follows: 1.PRMT5 mutations cause photorespiration-related genes to produce aberrant alternative splicing events.By RNA-seq,7018 genes were found in Arabidopsis Col-0 and prmt5-2 to undergo aberrantly alternative splicing,in which 5211 genes were retained in introns,and intron retention was significantly greater in prmt5-2.For Col-0,therefore,PRMT5 mutations affect the gene's alternative splicing,especially intron retention.From these mutated genes,we found that 20 photorespiration genes may be subject to alternative splicing.It was confirmed by PCR and transgene complementation experiments that 11 photorespiration related genes such as PGLP1 produced aberrant pre-mRNAs after PRMT5 mutation and the type of alternative splicing was intron retention.2.Possible functions of photorespiration-related genes that produce aberrant alternativ e splicing.Not only the alternative splicing increases the diversity of the encoded protein,but may also be degraded by nonsense-associated degradation pathways due to the generation of premature stop codons.We obtained a double mutant of upf1/prmt5-2 and upf3/prmt5-2 by crossing the mutants upf1 and upf3 of the nonsense-related degradation pathway-associated T-DNA with prmt5-2.Using these double mutants,we demonstrated that the aberrant alternative splicing generated by GLT1 and PGLP2 was up-regulated in the upf1/prmt5-2 and upf3/prmt5-2 expressions compared to prmt5-2,indicating that the two genes produced the aberrant transcripts degraded by the nonsense mediated decay pathway;whereas the alternative splicing generated by the other 9 genes showed little difference between double mutants and single mutants and mRNAs produced by alternative splicing may produce new proteins,but further experiments are needed to confirm.3.Effects of alternative splicing on the enzymatic activity of AGT.Since AGT produces aberrant transcripts in prmt5-2,which is not related to nonsense-associated degradation,and new proteins may be produced.According to the AGT sequence,AGT can produce three different mRNAs in prmt5-2,which are the open reading frame encoding AGT,the open reading frame of AGT and the first intron sequence;The open reading frame of AGT retained the first and eighth intron sequences;three different mRNAs recombinant expression vectors of AGT were constructed by means of recombinant expression and Col-0 and prmt5-2 were impregnated with floral dipping methods.Transgenic homozygous plants were obtained.The results showed that the overexpression of AGT open reading frame of containing two intron sequences increased the expression of AGT but decreased the activity of alanine and glutamate.The expression of AGT in the transgenic materials overexpressing the AGT open reading frame was greatly improved.The activity of glutamate was greatly enhanced in both Col-0 and prmt5-2 and the activity of alanine was enhanced in only prmt5-2.While the transgenic material overexpression of the AGT open reading frame of containing the first intron sequence increased the expression of AGT but had little effect on the activity of alanine and glutamate.It was demonstrated that the overexpression of AGT in the open reading frame can increase the enzyme activity of AGT,while the overexpression of AGT containing intron transcript sequences has little effect on enzyme activity and even inhibits the enzyme activity.4.The function regulated gene's alternative splicing by PRMT5 is similar between Rice and Arabidopsis.RNA-seq was performed by using OsPRMT5 gene editing materials.As a result,it was found that loss of function of OsPRMT5 also resulted in aberrant alternative splicing of many genes.2433 alternative splicing events in Zhonghua 11 were intron retention.There are 3267 intron retention events in OsPRMT5-cas9 82-35,and there are no significant differences in the other types of alternative splicing;among these genes,we found that rhythm,flowering delay,splicing-related genes and photorespiration-related genes produce similar alternative splicing events between the OsPRMT5 and AtPRMT5,suggesting PRMT5 mutation has the same features in intron retention between Arabidopsis and Rice.