The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns

Pessa, Heli K. J.; Ruokolainen, Annukka; Frilander, Mikko J.

doi:10.1261/rna.213906

Cited by 32 publications

(39 citation statements)

References 32 publications

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“…However, as snRNPs are present in large excess in the cell, it is unlikely that a 50% reduction in di-snRNP levels alone would cause the observed effects. Indeed, residual amounts (Ͻ20%) of U4atac snRNA are sufficient to support the splicing of endogenous pre-mRNAs at WT levels (25), suggesting that the reduction of a single spliceosomal component by 80 to 90% does not a priori lead to splicing inhibition.…”

Section: Discussionmentioning

confidence: 99%

“…Intriguingly, based on the migration behavior of the 48K protein on polyacrylamide gels, it was previously suggested that the protein may be posttranslationally modified (38). The excision of U12-type introns appears to be the ratelimiting step in the splicing of genes containing both types of introns (23,25). Therefore, the posttranslational modification of unique spliceosomal factors, such as 48K, may potentially allow for the rapid regulation of the U12-dependent spliceosome and thus affect the expression of genes with U12-type introns.…”

Section: Discussionmentioning

confidence: 99%

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

Turunen

Will

Grote

et al. 2008

Molecular and Cellular Biology

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Turunen

Will

Grote

et al. 2008

Molecular and Cellular Biology

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“…A key characteristic of U12-type introns is that they are removed more slowly than U2-type introns, suggesting they may serve as rate-limiting controls for the expression of their host genes (35,36). The slow processing rate of the U12-dependent spliceosome was suggested by König et al (23) to lead to the export of unspliced pre-mRNAs from the nucleus and entail the need for splicing activity in the cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

“…The subcellular localization of U1, U2, U6, U11, U12, U4atac, and U6atac snRNAs in several embryonic mouse tissues was analyzed by nonradioactive cRNA ISH on tissue sections. Sagittal sections of E15 NMRI mouse embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and serially sectioned at 5 m. cRNA probes were labeled with DIG by performing in vitro transcription of snRNA clones (5,36) in the presence of DIG RNA labeling mix (Roche). Sense probes were synthesized from the complementary strand of the probe templates.…”

Section: Methodsmentioning

confidence: 99%

Minor spliceosome components are predominantly localized in the nucleus

Pessa

Will

Meng³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Recently, it has been reported that there is a differential subcellular distribution of components of the minor U12-dependent and major U2-dependent spliceosome, and further that the minor spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and minor spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12-and U2-dependent spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major spliceosome, components of the U12-dependent spliceosome are localized predominantly in the nucleus.localization ͉ pre-mRNA splicing ͉ snRNA ͉ U12-dependent spliceosome P re-mRNA splicing is an essential step in the co/posttranscriptional processing of eukaryotic transcripts before their export from the nucleus. The low-abundance U12-dependent ''minor'' spliceosome exists in parallel with the U2-dependent ''major'' spliceosome in most multicellular eukaryotes (1). It catalyzes the removal of a rare class of introns (U12-type) that represent Ͻ1% of introns in mammals (2, 3). The U12-dependent spliceosome contains four unique snRNAs: U11, U12, U4atac, and U6atac, which are paralogs of U1, U2, U4, and U6 snRNAs of the U2-dependent spliceosome, respectively (4, 5). U5 snRNA, in contrast, is shared between the two spliceosomes. In addition to the snRNAs, both spliceosomes contain numerous snRNP and non-snRNP proteins (6, 7).The five snRNP components of the major spliceosome are predominantly localized in the nucleus, but the biogenesis of four of them involves a cytoplasmic step (8). That is, after transcription by RNA Pol II, the snRNAs U1, U2, U4, and U5, which all contain an Sm protein-binding site, are first exported into the cytoplasm. The Sm proteins subsequently bind, and the snRNA's cap is hypermethylated to 2,2,7-tri-methyl-guanosine (m 3 G) (9, 10). These processing steps are a prerequisite for their reimport into the nucleus (11). In contrast to these so-called Sm-class snRNAs, U6 snRNA is transcribed by RNA pol III, acquires Sm-like proteins (Lsm2-8) (12) and a ␥-monomethyl phosphate cap, and does not leave the nucleus. The minor snRNAs U11, U12, and U4atac are also Sm-class snRNAs, containing an Sm-binding site that is also bound by Sm proteins and an m 3 G cap (5, 13), whereas U6atac is transcribed by RNA pol III, possesses a ␥-monomethyl phosphate cap (5), and is assembled with Lsm proteins (14). After nuclear import, spliceosomal snRNPs first pass through Cajal bodies, where they undergo further modifications and assembly (15, 16) and then subsequentl...

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“…11,12 Another difference is that minor introns have been reported to be spliced more slowly, both in vitro 13,14 and also in vivo. [15][16][17][18] The slower splicing in vivo has been inferred from the elevated levels of unspliced U12-type introns in the cellular steady-state RNA pool. On average, U12-type introns show approximately 2-fold higher retention levels compared to the U2-type introns in the same gene.…”

Section: Introductionmentioning

confidence: 99%

Regulation of gene expression through inefficient splicing of U12-type introns

Niemelä

Frilander

2014

RNA Biology

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U 12-type introns are a rare class of nuclear introns that are removed by a dedicated U12-dependent spliceosome and are thought to regulate the expression of their target genes owing through their slower splicing reaction. Recent genome-wide studies on the splicing of U12-type introns are now providing new insights on the biological significance of this parallel splicing machinery. The new studies cover multiple different organisms and experimental systems, including human patient cells with mutations in the components of the minor spliceosome, zebrafish with similar mutations and various experimentally manipulated human cells and Arabidopsis plants.Here, we will discuss the potential implications of these studies on the understanding of the mechanism and regulation of the minor spliceosome, as well as their medical implications.

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The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns

Cited by 32 publications

References 32 publications

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

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

Minor spliceosome components are predominantly localized in the nucleus

Regulation of gene expression through inefficient splicing of U12-type introns

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