Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins

Abstract

During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.

Extending the critical regions for mutations in the non‐coding gene RNU4ATAC in another patient with Roifman Syndrome

“…There are discrepant results for NR_023343.1:n.116A which, depending on the publication, either had or had not been judged as part of the Sm protein‐binding site. The core region, essential for splicing, is so far defined from NR_023343.1:n.117 to NR_023343.1:n.124 2, 3, 13. Regarding the clinical data obtained in the study presented here, NR_023343.1:n.116 also belongs to the critical region important for splicing, which has to be confirmed in further functional studies (Figure 1).…”

“…Both variant‐positions are highly conserved. The paternally inherited variant n.13C>T is located in an element of major importance for splicing at the stem II, the maternally inherited variant n.116A>C is located in an element of variable importance for splicing at the transition of 3′stem‐loop to the Sm protein‐binding site2, 5, 12, 13 …”

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

“…One half of the beads were run on 12% SDS-PAGE and subjected to Western blotting and Ponceau S staining. Antibodies used include anti-Sm Y12 (Lab Vision Inc., Fremont, CA), anti-U2B″ (Helen Salz, Case Western Reserve University, Cleveland, OH), and an antibody to the 60 kD protein associated with the U4/U6 snRNP (Nottrott et al 2002). The other half of the reaction beads were resuspended in 100 μl DEPC-treated water and extracted twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1).…”

“…7a). We also tested whether the coilin fragments would recover a 60 kDa protein known to associate with the U4/U6 snRNPs (Nottrott et al 2002). Whereas no signal was observed in the GST and GST-C156 lanes, both the larger coilin fragment (GST-C214) and GST-SMN recovered this protein (Fig.…”

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…only RNase T1 (which cuts exclusively 3 0 to G) lead to very intense RNA product ions in gas-phase fragmentation in the mass spectrometer. These suppress the fragment ions derived from the cross-linked peptide, so that the peptide sequence can hardly be determined under these conditions in the mass spectrometer [38].…”