Expression and Purification of Serine/Arginine-Rich Splicing Factor 1 from Escherichia coli Expression System

Serine/arginine-rich splicing factor 1(SRSF1), as a prototype member of the highly conserved serine/arginine family of RNA binding proteins, plays an important role in mRNA alternative splicing, stabilization, nuclear export, and translation. Here, the expression system was established to purify full-length human SRSF1 from Escherichia coli(E. coli). The SRSF1 coding sequence was amplified by polymerase chain reaction(PCR) and inserted into the pET-28 a-ppSUMO vector with His-tag to construct a recombinant plasmid His-SUMO-SRSF1. Then the plasmid was transformed into BL21(DE3) competent cells for expression. After purification by affinity chromatography and cleavage of His-SUMO moiety, a highly purified SRSF1 with a molecular weight of around 28 kg/mol was obtained. The protein was analyzed by sizing chromatography and it was found that SRSF1 would form a polymer structure in the solution. According to Expasy bioinformatics analysis, SRSF1 is extremely unstable. Purification of full-length SRSF1 protein provides an opportunity to study mRNA splicing in vitro.

Cassava Me RS40 is required for the regulation of plant salt tolerance

Soil salinity affects the expression of serine/arginine-rich(SR) genes and isoforms by alternative splicing, which in turn regulates the adaptation of plants to stress.We previously identified the cassava spliceosomal component 35 like(SCL) and SR subfamilies, belonging to the SR protein family, which are extensively involved in responses to abiotic stresses.However, the post-transcriptional regulatory mechanism of cassava arginine/serine-rich(RS) subfamily in response to salt stress remains to be explored.In the current study, we identified 37 genes of the RS subfamily from 11 plant species and systematically investigated the transcript levels of the RS40 and RS31 genes under diverse abiotic stress conditions.Subsequently, an analysis of the conserved protein domains revealed that plant RS subfamily genes were likely to preserve their conserved molecular functions and played critical functional roles in responses to abiotic stresses.Importantly, we found that overexpression of MeRS40 in Arabidopsis enhanced salt tolerance by maintaining reactive oxygen species homeostasis and up-regulating the salt-responsive genes.However,overexpression of MeRS40 gene in cassava reduced salt tolerance due to the depression of its endogenous gene expression by negative autoregulation of its own pre-mRNA.Moreover, the MeRS40 protein interacted with MeU1-70Ks(MeU1-70Ka and MeU1-70Kb) in vivo and in vitro, respectively.Therefore, our findings highlight the critical role of cassava SR proteins in responses to salt stress in plants.

Specific Regulation of m^(6)A by SRSF7 Promotes the Progression of Glioblastoma

Serine/arginine-rich splicing factor 7(SRSF7),a known splicing factor,has been revealed to play oncogenic roles in multiple cancers.However,the mechanisms underlying its oncogenic roles have not been well addressed.Here,based on N6-methyladenosine(m^(6)A)co-methylation network analysis across diverse cell lines,we find that the gene expression of SRSF7 is positively correlated with glioblastoma(GBM)cell-specific m^(6)A methylation.We then indicate that SRSF7 is a novel m^(6)A regulator,which specifically facilitates the m^(6)A methylation near its binding sites on the mRNAs involved in cell proliferation and migration,through recruiting the methyltransferase complex.Moreover,SRSF7 promotes the proliferation and migration of GBM cells largely dependent on the presence of the m^(6)A methyltransferase.The two m^(6)A sites on the mRNA for PDZ-binding kinase(PBK)are regulated by SRSF7 and partially mediate the effects of SRSF7 in GBM cells through recognition by insulin-like growth factor 2 mRNA-binding protein 2(IGF2BP2).Together,our discovery reveals a novel role of SRSF7 in regulating m^(6)A and validates the presence and functional importance of temporal-and spatial-specific regulation of m^(6)A mediated by RNA-binding proteins(RBPs).

Translation rate underpins specific targeting of N-terminal transmembrane proteins to mitochondria

Protein biogenesis is a complex process,and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles.To direct targeting,organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery.However,the situation is confusing for transmembrane domain(TMD)-containing signalanchored(SA)proteins of various organelles because TMDs function as an endoplasmic reticulum(ER)targeting signal.Although ER targeting of SA proteins is well understood,how they are targeted to mitochondria and chloroplasts remains elusive.Here,we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts.Mitochondrial targeting requires multiple motifs around and within TMDs:a basic residue and an arginine-rich region flanking the N-and C-termini of TMDs,respectively,and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner.These motifs play a role in slowing down the elongation speed during translation,thereby ensuring mitochondrial targeting in a cotranslational manner.By contrast,the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post-translational manner.