Identification of RNAs that can accept assembly of an Sm-protein ring

Sm-ring assembly is important for the biogenesis, stability, and function of uridine-rich small nuclear RNAs (U snRNAs) involved in pre-mRNA splicing and histone pre-mRNA 3' end processing. Assembly of Sm-rings occurs in the cytoplasm and is dependent on a specific sequence and structure motif (Sm-site), ATP, and the Survival motor neuron (SMN) protein complex. The following study informatically investigates the occurence of Sm-sites within the mouse and human transcriptomes and biochemically assesses whether these sites can accept Sm-rings. Sm-sites were found on snRNAs, but are highly prevalent in the 3' untranslated regions (3'UTR) of long mRNAs. RNA immunoprecipitation experiments confirm that Sm-site containing mRNAs associate with Sm-proteins in the cytoplasm. Established Sm-ring assembly assays were modified to identify polyA-RNAs that specifically associate with Sm-proteins in an ATP-dependent manner, modeling newly assembled Sm-rings. Sm-rings were then specifically assembled onto candidate Sm-site containing mRNAs in an ATP and Sm-site dependent manner. mRNAs containing Sm-sites are down-regulated in models of SMA, suggesting reduced Sm-ring assembly on these mRNAs may contribute to the mechanism of SMA pathogenesis. Together, this study establishes that Sm-site containing mRNAs can accept Sm-rings and identifies a novel mechanism for Sm-proteins in regulation of cytoplasmic mRNAs. Overall design: To identify human and mouse RNAs that can accept Sm-rings, four RNA-sequencing library conditions were compared. 1) a polyA-RNA sequencing library obtained by oligo-dT enrichment from human S3-iNPC or mouse NSC-34 cell extracts. 2) an anti-Sm RNA immunoprecipitation from cytoplasmic cell extracts isolated from human S3-iNPC or mouse NSC-34 cells. 3) a modified Sm-ring assembly condition in which an anti-Sm-RIP was performed following incubation of human polyA-RNA within a mouse cytoplasmic cell extract, but in the absence of ATP (also performed in the opposite direction). 4) lastly, a modified Sm-ring assembly condition in which an anti-Sm-RIP was performed following incubation of human polyA-RNA within a mouse cytoplasmic cell extract, but in the presence of ATP (also performed in the opposite direction). Each condition was performed in triplicate and sequencing libraries were generated from isolated RNA following Takara SMARTer Stranded Total RNA-Seq Kit v3-Pico Input Mammalian (Takara 634451).