Genetic Analysis Of C.elegans Splicing Factor Mutant Mfap-1(n4564 N5214) Suppressor Sftb-2(mac30)

Background: RNA splicing is a key regulatory process in eukaryotic gene expression.It removes non-coding introns of pre-m RNA(pre-m RNA)and connects exons containing codons and regulatory sequences through complex splicing molecular machinery,thereby producing mature m RNA.Alternative splicing can use different mechanisms to generate variable m RNAs from the same pre-m RNAs,which affects m RNA stability,complexity of gene expression,and protein diversity in the organism.RNA splicing is executed by coordinated actions of multiple splicing factors.We previously isolated a mutation,n4564n5214,in the splicing factor gene mfap-1 as a suppressor of the uncoordinated phenotype of unc-93(e1500)mutants.This mutation causes a temperature sensitive(ts)lethal phenotype(lethal at 22.5 °C or higher temperatures).We further isolated a mutation in the splicing factor gene sftb-2,mac30,as a suppressor of the ts-lethal phenotype of mfap-1(n4564n5214)mutants.sftb-2 encodes the C.elegans ortholog of SF3B2,a key component of the U2 sn RNP-interacting splicing complex SF3 B.Mutations in SF3B2 or expression level changes of SF3B2 have been associated with cancers.Objectives: To explore the genetic mechanism of sftb-2 mutation suppressing the temperature-sensitive lethal phenotype of mfap-1mutation,and to perform genetic analysis of sftb-2.Methods:(1)Use temperature-shift experiments to determine the developmental stage of mutant strains affected by high temperatures.(2)Use transcriptome and RT-PCR analyses to understand how sftb-2 and mfap-1 affect gene expression and RNA splicing.(3)Use locomotion assay to analyze how sftb-2 interacts with mfap-1 or uaf-1 to affect the behavior of unc-93(e1500)mutants.Results:(1)By analyzing how different temperatures affect egg-hatching and larval growth,we found that high temperatures(22.5 °C and 25 °C)primarily affected the reproduction and egg development of mfap-1(n4564 n5214)mutants.(2)sftb-2(mac30)suppressed the defective expression and splicing of certain genes in mfap-1(n4564 n5214)mutants.(3)sftb-2(mac30)suppressed the effects mfap-1(n4564 n5214)and uaf-1(n4588)on the behavior of unc-93(e1500)mutants.Conclusions: sftb-2 and mfap-1 potentially interact to affect gene expression and alternative splicing.sftb-2(mac30)might affect the behavior of unc-93(e1500)mutants by interacting with mfap-1 and uaf-1.