U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro

Abstract

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5′ splice sites at exon/intron boundaries. U1 snRNAs associate with 5′ splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the ‘Smith antigen’, or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and (IPs) precipitations revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the hU1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs.

Transcriptional analysis supports the expression of human snRNA variants and reveals U2 snRNA homeostasis by an abundant U2 variant

“…Eight of the transcribed U1 genes have mature sequences identical to the canonical U1 snRNA gene, but variation in the flanking sequences and mapping reads to multiple genomic loci allowed some to be identified as transcribed with high confidence. We identified transcribed genes with sequence variation that match 6 of 8 previously identified variant U1 transcripts (O'Reilly et al, 2013;Somarelli et al, 2014). We also identified two transcribed genes from the variant U1 cluster that were previously examined and thought not to be transcribed.…”

“…The compensatory increase in canonical U2-1 upon U2-2 knockout might have been predicted when considering that introducing human U1 to mouse cells did not increase overall U1 levels (Mangin et al, 1985), and in differentiating cells canonical and variant U1 snRNAs are inversely expressed (Vazquez-Arango et al, 2016). It has also been observed that variant U1 sequences are able to bind multiple components of U1 snRNP but are out-competed by the canonical U1 snRNA (Somarelli et al, 2014). In human tissues where the ratio of U2-2 to U2-1 varies, U2-1 expression might be modulated by promoter accessibility or the abundancy of transcription factors.…”

Insights into the U1 small nuclear ribonucleoprotein complex superfamily

“…Like the vU1 snRNAs previously described in other species, this group of human vU1 snRNAs is primarily expressed in human stem and carcinoma cells, contain a trimethyl cap structure at the 5′ end and are packaged into Sm‐containing complexes. With regards the core proteins, including U1‐C, U1‐A, and U1‐70K, only vU1 snRNAs with the highest identity to U1 are capable of associating with all of these proteins in vivo in general . Gel shift assays using recombinant core proteins with in vitro transcribed U1/vU1 snRNA demonstrate that most of the vU1 snRNAs analyzed display a range of binding capabilities, with varying affinities, for the U1‐specific core proteins when compared to the U1 snRNA.…”

“… Several of the dispersed vU1 genes have no homology in their flanking regions to each other or to the U1 snRNA genes and are typically flanked by repeat elements or stretches of adenines or thymines . While the lack of any recognizable promoter sequences and 3′ end processing elements would suggest that such U1 copies are transcriptionally inactive, vU1 snRNAs matching these genes copies have previously been detected in human cells . Although no known function(s) has been assigned to these vU1 copies, it does suggest, at least, that U1 snRNA expression can be controlled by mechanisms other than that which has been previously described for the RNU1.1‐4 U1 genes .…”

Cutting a Long Intron Short: Recursive Splicing and Its Implications

“…We have also come to find out that the human genome encodes a large number of “variant” U1 snRNAs (Kyriakopoulou et al, 2006; O'Reilly et al, 2013). Their expression is markedly higher in primary, embryonic, and pluripotent cells (O'Reilly et al, 2013; Kelly et al, 2015; Vazquez-Arango et al, 2016) and they are able to form proper RNPs in vitro (Somarelli et al, 2014). In endothelial cells, the repertoire of expressed variant U1, together with the minor spliceosome (Turunen et al, 2013), would suffice for the recognition of the vast majority of all non-canonical RS donor dinucleotides recorded (Kelly et al, 2015).…”