Proximity of the U12 snRNA with both the 5′ Splice Site and the Branch Point during Early Stages of Spliceosome Assembly

Abstract

U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5 end of the U12 snRNA and the ؊2 position upstream of the 5 splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5 exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5 exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5 splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5 exon sequences near 5 splice site and the branch point sequence, suggesting that the 5 splice site and branch point sequences are separated by <40 to 50 Å in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.During pre-mRNA splicing, the 5Ј splice site (5Јss) and the branch point sequence (BPS), located at the opposite ends of each intron, must come into close proximity to facilitate the chemistry of the first catalytic step (4). In metazoan cells, two different types of introns, namely, the U2 type and U12 type, are recognized by their different 5Јss and BPS sequences. The U2-type introns comprise Ͼ99.5% of all spliceosomal introns and contain relatively poorly conserved 5Јss and BPS regions. In contrast, these sequences are highly conserved in the lowabundance U12-type introns (13, 47).Distinct U2-and U12-dependent spliceosomes facilitate the removal of the two different intron types. These two spliceosomes differ in their composition of small nuclear ribonucleoproteins (snRNPs), but their overall assembly pathways and catalytic mechanisms are similar. The U1, U2, U4, and U6 small nuclear RNAs (snRNAs) are specific to the U2-dependent spliceosome, whereas U11, U12, U4atac, and U6atac snRNAs are functionally analogous components in the U12-dependent spliceosome. Both spliceosomes utilize the U5 snRNA (42,56).A widely accepted paradigm of spliceosome assembly states that snRNPs and protein components associate with the premRNA in a stepwise manner (3,4,16). In the U2-dependent spliceosome, this process starts with intron recognition in complex E (the commitment complex in yeast), in which the 5Јss is recognized by the U1 snRNP, whereas the BPS, polypyrimidine tract (PPT), and 3Ј splice site (3Јss) are recognized by the protein factors SF1/BBP, U2AF65, and U2AF35, respectively (1,36,37,44,68,75). In the first ATP-dependent stage (complex A, or prespliceosome), SF1/BBP at the BPS is replaced by the U2 snRNP (43, 74). A spliceosome (complex B) is formed upon entry of the U4/U6.U5 tri-snRNP (20,24). Additional rearrangements of RNA-RNA interactions (39, 50), and changes in protein comp...

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

“…This spatial arrangement of the proteins (Fig. 7) might also enable the spliceosome to achieve the right catalytic orientation for splicing, 85,111 as observed in the recent near atomic structure of the yeast spliceosome where much of the catalytic center is shaped by protein components. 59 Finally, this arrangement might also aid in the attack of BPA at the 5 0 splice site in a diffusion limited regime by limiting pre-mRNA motions in the local vicinity of the spliceosomal proteins.…”

“…The dotted lines show the spacing between the nucleotides with a distance of 54.3 A and a close agreement with earlier biochemical studies. 85 The boxes show the exposed U12 snRNA regions where it interacts with 5 0 splice site and branch point adenosine of pre-mRNA. The pre-mRNA (yellow strand) that interacts with U12 snRNA and forms a duplex (boxed region) is modeled on a U2 snRNA: pre-mRNA crystal structure duplex, the rest of the strand is modeled to show a speculative path.…”

“…This prompts us to speculate that the SF3b open form acts as a "scaffold" for the 5 0 end of U12 snRNA, which is topologically consistent with the biochemical data. 85 Furthermore, it is important to note that the U12 snRNA: premRNA structure was modeled purely guided by the structural constraints provided by the spatial positioning of proteins SF3b49, SF3b145 as well as p14 (interaction with branch point duplex). Therefore, these proteins, especially SF3b49 and SF3b145, can possibly act as RNA chaperones 96,97 for the U12 snRNA to take up the right tertiary structure, analogous to that observed in the case of ribosomal proteins for rRNA.…”

“…Previously, a combination of deletion analysis and RNA crosslinking studies have reported that the U12 snRNA interacts with both the 5 0 splice site and branch point sequence, within an estimated distance of about 40 to 50 A between nucleotides 4 and 16 of U12 snRNA. 7,85 We measured the distance between these nucleotides in our U12 snRNA structure model. Remarkably, it has a close agreement (»54.3 A ) with the earlier observation (Fig.…”

“…Hence, we modeled the branch point duplex (BPD) containing U12 snRNA and pre-mRNA, including the BPA, using an available crystal structure of BPD (PDB ID: 1I9X) 84 and prior knowledge of U12 snRNA: pre-mRNA base pairing. 85 The information on the recognition of BPA by p14 was collated from multiple sources 9,30 and the p14-BPD complex structure was modeled using a restrained docking procedure (see Materials and Methods). Further, we found that the p14-BPD docked complex structures fitted snugly into the groove above p14 density surface, when fitted in the U11/U12 di-snRNP cryo-EM map.…”