U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

Abstract

Formation of catalytic core of the U12-dependent spliceosome involves U6atac and U12 interaction with the 5′ splice site and branch site regions of a U12-dependent intron, respectively. Beyond the formation of intermolecular helix I region between U6atac and U12 snRNAs, several other regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that the 3′ stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity. Here, we show a detailed structure-function analysis and requirement of a substructure of U6atac 3′ stem-loop in U12-dependent in vivo splicing. We show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3′ stem-loop of U6atac. By using a binary splice site mutation suppressor assay we demonstrate that p65 protein-binding apical stem-loop of U12 snRNA can be replaced by this U6atac distal 3′ stem-loop. Furthermore, we tested the compatibility of the U6atac 3′ end from phylogenetically distant species in a human U6atac background, to establish the evolutionary relatedness of these structures and in vivo function. In summary, we demonstrate that RNA-RNA and RNA-protein interactions in the minor spliceosome are highly plastic as compared to the major spliceosome.

Mutations in the U11/U12-65K protein associated with isolated growth hormone deficiency lead to structural destabilization and impaired binding of U12 snRNA

“…The model described above does not account for the potential role of the disrupted 65K-U6atac interaction in IGHD. Consistent with a recent report documenting the interaction between 65K C-RRM and the distal stem-loop of U6atac snRNA (Singh et al 2016), our biochemical analysis confirmed that the 65K C-RRM can indeed bind to U6atac, but with approximately threefold reduced affinity compared to U12 snRNA. Furthermore, we documented a similar additional threefold reduction in the U6atac snRNA binding with the P474T mutant and a complete loss of binding with R502X.…”

“…The 3 ′ end of U6atac, which includes the 65K-binding stem-loop, has been demonstrated to play a role in guiding U6atac snRNA to the minor spliceosome (Dietrich et al 2009). Although U6atac snRNA can be pulled down with 65K from HeLa cell lysate (Singh et al 2016), it is not known if the 65K protein associates with the U4atac/U6atac.U5 tri-snRNP or whether, in addition to the A complex, it is present in later spliceosomal complexes. Interestingly, analysis of a zebrafish 65K mutant revealed an accumulation of a slow-migrating complex containing U12, U5, and U6atac snRNAs, leading the authors to suggest an additional role for 65K in the later stages of the splicing process, such as spliceosome disassembly or recycling (Markmiller et al 2014).…”

“…To address the impact of IGHD-causing mutations within the 65K protein on binding of the U12 or U6atac snRNAs in vitro, we carried out RNA-binding assays using recombinant wild-type (WT) or mutant (P474T or R502X) 65K C-RRM and 32 P-labeled short RNA hairpin substrates described earlier ( Fig. 1A; Benecke et al 2005;Singh et al 2016). As the untagged P474T-mutant RRM is stable in soluble form under RNA-binding conditions for only a short time after the tag cleavage, the in vitro binding assays were carried out using tagged proteins, while untagged proteins were used for all other experiments (Supplemental Fig.…”

“…It contains two RNA recognition motifs (RRMs), of which the carboxy-terminal RRM (C-RRM) is used to bind to the 3 ′ terminal stem-loop of the U12 snRNA (Benecke et al 2005). Additionally, a recent work demonstrated that the C-RRM also interacts with the nearidentical 3 ′ terminal stem-loop of the U6atac snRNA (Singh et al 2016). Within the U11/U12 di-snRNP the amino-terminal part of the 65K protein in turn interacts with the U11 snRNP-specific 59K protein (Benecke et al 2005), which itself interacts with U11-48K, a protein that participates in recognition of the U12-type 5 ′ splice site with U11 snRNA (Turunen et al 2008;Tidow et al 2009).…”

Early developmental arrest and impaired gastrointestinal homeostasis in U12-dependent splicing-defective Rnpc3-deficient mice

“…The N-terminal portion of the 65K protein binds to the 59K protein and its C-terminal RNA binding domain binds to U12 snRNA Singh et al 2016) (Supplemental Fig. S1B).…”