The U11-48K Protein Contacts the 5′ Splice Site of U12-Type Introns and the U11-59K Protein

Abstract

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5 splice sites (5ss). This interaction did not require branch point recognition and was sensitive to 5ss mutations, suggesting that 48K interacts with the 5ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5ss, as well as functionally important interactions within the U11/U12 di-snRNP.Most metazoans and some unicellular eukaryotes contain two distinct spliceosomes (reference 28 and references therein). The majority of introns are excised by the major, U2-dependent spliceosome, while a subset (Ͻ0.5%) with highly conserved 5Ј splice sites (5Јss) and branch point sequences (BPS) are removed by the minor, U12-dependent spliceosome (for a review, see reference 24). Both spliceosomes consist of five small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins. The two spliceosomes share the U5 snRNP, while the remaining four snRNPs in each spliceosome are distinct but functionally analogous, with U11, U12, U4atac, and U6atac of the minor spliceosome being the counterparts of U1, U2, U4, and U6 in the major spliceosome, respectively (12,14,16,34,35,42).Intron recognition is achieved via multiple, dynamic RNAand protein-mediated interactions. RNA-RNA interactions in the two spliceosomes are highly analogous. In the major spliceosome, the first assembly step (generating the E complex) involves the recognition of the 5Јss by U1, while the nonsnRNP protein factors SF1, U2AF65, and U2AF35 bind to the BPS, the polypyrimidine tract, and the 3Јss, respectively (for a review, see reference 26). During this stage, U1 base pairs with the pre-mRNA's 5Јss, while U1-associated proteins facilitate 5Јss recognition or stabilize the U1-5Јss complex (for a review, see reference 37). During prespliceosome (A complex) formation, U2 associates stably with the BPS (17), while non-snRNP proteins bridge U1 and U2 snRNPs (40, 41). The U4/U6/U5 tri-snRNP then joins the spliceosome (generating the B complex), which triggers the displacement of U1 from the 5Јss by U6 and additional rearrangements that lead to catalytic core formation (for a review, see references 22 and 33).The overall assembly pathway of the minor...

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

“…[9][10][11][12][13] Earlier, we have described such a negative feedback loop in two genes encoding for core protein components that are specific to the U12-dependent spliceosome (also called minor spliceosome). 12 Both of these proteins, U11-48K and U11/U12-65K (also known as RNPC3), are integral components of the U11/ U12 di-snRNP, [14][15][16] which recognizes the 5 0 splice site (5 0 ss) and the branch point sequence (BPS) of U12-type introns. [17][18][19] Both genes contain a novel splicing regulatory element denoted USSE (U11 snRNP-binding splicing enhancer), which is composed of a tandem duplication of 5 0 ss sequences of U12-type introns that are, however, not used for splicing.…”

Minor class splicing shapes the zebrafish transcriptome during development

“…Activation of cryptic splice sites also occurs in human cells upon knockdown of the U11/U12 48-kDa protein with RNAi (30). To determine whether cryptic splice sites are activated in clbn, we sequenced the spliced transcripts obtained by RT-PCR for sms, mapk3, and mapk12 mRNAs (Fig.…”

HnRNPH1/H2, U1 snRNP, and U11 snRNP cooperate to regulate the stability of the U11-48K pre-mRNA

“…Site-specific protein-RNA crosslinks were formed by irradiating samples with 254-nm UV light (1 J/cm 2 ) on ice. For IP, samples were denatured with 0.2% SDS at 95°C, incubated with antibodies, and washed according to the method described by Turunen et al (2008). Proteins were separated by SDS-PAGE and visualized by autoradiography on phosphoimager plates, which were scanned with Fuji FLA-5000 scanner, and the obtained images were quantified using AIDA image analyzer software.…”

“…Site-specifically labeled RNAs with similar sequences were constructed essentially according to the method previously described by Turunen et al (2008). Briefly, the 3 ′ portion of the construct (starting with the last nucleotide of exon 4i) was produced by in vitro transcription with T7 RNA polymerase, dephoshorylated, and labeled at the 5 ′ end with [ 32 P]-phosphate.…”