Identification Of A Novel Splicing Variant And Preliminary Study Of SMG9

In the process of evolution,there are many mRNA quality survilliance mechanisms to protect the veracity of the genetic message in eukaryotes.Nonsense-mediated mRNA decay(NMD)scrutinizes mRNA quality as a translation-dependent manner.NMD is not only degrades the aberrant mRNAs which contain premature termination codon(PTC),it can also dynamically adjust transcriptomes and proteomes in mammalian cells in order to vary physiological conditions.Alternative splicing(AS)can make a gene to produce different isoforms during pre-mRNA splicing.However,not all spliced products can be translated into a protein and exert its normal functions.A part of them may contain a PTC,which would be decayed by NMD.SMG9(nonsense mediated mRNA decay factor)is an important factor of NMD,which forms a complex with SMG1 and SMG8 to regulate the phosphorylation of UPFI.In the present study,we found that SMG9 underwent the alternative splicing and produced a new isoform.We demonstrated the novel isoform of SMG9,SMG9b,and analyzed its expression and regulation.SMG9b is involved in NMD pathway and controls the substrate of NMD.Furthermore,NMD regulates the expression level of SMG9b in turn.This finding not only made us to understand the NMD mechanism and the process of feedback regulation more in-depth,but also provided a theoretical basis for the molecular mechanism of NMD.The main results are as follows:(1)The identification of SMG9 isoform.The full-length of SMG9b CDS is 927bp,and can encode a protein containing 308 amino acids.We record the reported SMG9 as SMG9a,and the novel splice isoform is denoted as SMG9b.(2)The expression of the SMG9b was detected by real-time quantitative PCR and Western blotting.The results suggested that SMG9b mRNA expression was higher than SMG9a,the protein expression level of SMG9b,however,was lower than SMG9a.(3)We found that SMG9a and SMG9b may interact with each other from overexpression and interference assay.That is,when SMG9a expression level increased,SMG9b expression level also raised.In addition,when SMG9a reduced,meanwhile,SMG9b decreased,and vice versa.(4)We found that miR-4651 can target SMG5 CDS by bioinformatics software prediction.The Q-PCR result showed that miR-4651 could suppress the expression of SMG9.(5)Overexpression of SMG9b reduced the expression of NMD substrate genes,which indicated that SMG9b could take part in NMD pathway.Interference of SMG9a specifically together with over-expression of SMG9b caused a decrease of NMD substrates,indicating that SMG9b would play a role in the NMD pathway directly.The interference of UPF1 can reduce the expression of SMG9b,which demonstrated that SMG9b is one of substrates of NMD.In general,although SMG9b shares a similar function with SMG9a in NMD,its expression regulation pattern seems to be more complex.SMG9b participates in NMD pathway to regulate the efficiency of NMD.On the other hand,it is also one of substrates of the NMD.These findings make the negative feedback regulation in NMD more noticeable.