Chromosomal instability by mutations in the novel minor spliceosome component CENATAC

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12‐dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT‐AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT‐AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.     

Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae

Structural and functional analysis of proteins involved in pre‐mRNA splicing is challenging because of the complexity of the splicing machinery, known as the spliceosome. Bioinformatic, proteomic, and biochemical analyses have identified a minimal spliceosome in the red alga Cyanidioschyzon merolae. This spliceosome consists of only 40 core proteins, compared to ～70 in S. cerevisiae (yeast) and ～150 in humans. We report the X‐ray crystallographic analysis of C. merolae Snu13 (CmSnu13), a key component of the assembling spliceosome, and present evidence for conservation of Snu13 function in this algal splicing pathway. The near identity of CmSnu13's three‐dimensional structure to yeast and human Snu13 suggests that C. merolae should be an excellent model system for investigating the structure and function of the conserved core of the spliceosome.     

39 In-silico study of the arrangement of the snRNPs within the native spliceosome

The elaborate process of transforming the information coded in the DNA to protein molecules is performed by several large and intricate molecular machines: RNA polymerase II transcribes the coded genes to pre-mRNAs, the spliceosome processes the pre-mRNAs, eliminating noncoding introns and producing functional mRNAs, and the ribosome translates the genetic code embedded in the mRNAs and catalyzes the synthesis of proteins. The spliceosome is a huge mega-Dalton ribonucleoprotein (RNP) assembly. Electron microscopy structures of the native spliceosome and of several spliceosomal subcomplexes, such as the spliceosomal U snRNPs, are available but the spatial arrangement of the latter within the native spliceosome is not known. We developed fitEM2EM computational tools (Frankenstein et al., 2008), that match and dock low resolution structures. Next, we represented each spliceosomal subcomplex by an ensemble of normal-modes conformers and designed a new “conformer selection” procedure that efficiently fitted the thousands of conformers into the native spliceosome envelope. Despite the low resolution limitations, we obtained only one model that complies with the available biochemical data. Our model localizes the five small nuclear RNPs (snRNPs), mostly within the large subunit of the native spliceosome, requiring only minor conformation changes. The remaining free volume presumably accommodates additional spliceosomal components. Moreover, the ample free volume suggests that structural modulations of the snRNPs can be tolerated while keeping the integrity of the spliceosome assembly. The constituents of the active core of the spliceosome are juxtaposed in our model, forming a continuous surface deep within the large spliceosomal cavity. This cavity emerges as the site of mRNA binding and splicing; its depth provides a sheltered environment for the splicing reaction (Frankenstein et al., 2012). To experimentally localize U snRNPs within the native spliceosome and validate the model, we use gold nanoclusters of 1.5 nm in diameter, covalently attached to antisense oligodeoxynucleotides, each complementary to one of the spliceosomal U snRNAs.     

Molecular cloning, genetic mapping, and expression of the mouse Sf3b1 (SAP155) gene for the U2 snRNP component of spliceosome

SAP155, a subunit of the U2 snRNP, is essential for prespliceosome assembly and splicing catalysis of the major spliceosome. Moreover, the protein has been identified in the minor (U12-dependent) spliceosome. These facts strongly suggest that SAP155 is shared by two distinct complexes owing to its importance in the removal of any type of intron. Here we have isolated a cDNA encoding the 146-kDa mouse homolog, designated Sf3b1. The amino acid sequence of Sf3b1 is very highly conserved among homologs from Schizosaccharomyces pombe (52.4% identity) to human (99.6%), and the C-terminal 825 residues of these Sf3b1 homologs show even higher identities. This C-terminal region shows significant similarity to the PR65 subunit of protein phosphatase 2A, which is composed of 15 tandem repeats of a 39 amino acid sequence. Mouse genome analyses showed Sf3b1 to be a single-copy gene mapping to the central part of Chromosome (Chr) 1. Northern blot analysis and whole mount in situ hybridization revealed Sf3b1 to be ubiquitously expressed in a variety of adult tissues and mid-gestation embryos. Received: 14 June 2000 / Accepted: 19 October 2000     

Evolutionary dynamics of U12-type spliceosomal introns

Background  

Many multicellular eukaryotes have two types of spliceosomes for the removal of introns from messenger RNA precursors. The major (U2) spliceosome processes the vast majority of introns, referred to as U2-type introns, while the minor (U12) spliceosome removes a small fraction (less than 0.5%) of introns, referred to as U12-type introns. U12-type introns have distinct sequence elements and usually occur together in genes with U2-type introns. A phylogenetic distribution of U12-type introns shows that the minor splicing pathway appeared very early in eukaryotic evolution and has been lost repeatedly.     

Computational screen for spliceosomal RNA genes aids in defining the phylogenetic distribution of major and minor spliceosomal components

The RNA molecules of the spliceosome are critical for specificity and catalysis during splicing of eukaryotic pre-mRNA. In order to examine the evolution and phylogenetic distribution of these RNAs, we analyzed 149 eukaryotic genomes representing a broad range of phylogenetic groups. RNAs were predicted using high-sensitivity local alignment methods and profile HMMs in combination with covariance models. The results provide the most comprehensive view so far of the phylogenetic distribution of spliceosomal RNAs. RNAs were predicted in many phylogenetic groups where these RNA were not previously reported. Examples are RNAs of the major (U2-type) spliceosome in all fungal lineages, in lower metazoa and many protozoa. We also identified the minor (U12-type) spliceosomal U11 and U6atac RNAs in Acanthamoeba castellanii, where U12 spliceosomal RNA as well as minor introns were reported recently. In addition, minor-spliceosome-specific RNAs were identified in a number of phylogenetic groups where previously such RNAs were not observed, including the nematode Trichinella spiralis, the slime mold Physarum polycephalum and the fungal lineages Zygomycota and Chytridiomycota. The detailed map of the distribution of the U12-type RNA genes supports an early origin of the minor spliceosome and points to a number of occasions during evolution where it was lost.     

The spliceosomal PRP19 complex of trypanosomes

In trypanosomes, mRNAs are processed by spliced leader (SL) trans splicing, in which a capped SL, derived from SL RNA, is spliced onto the 5′ end of each mRNA. This process is mediated by the spliceosome, a large and dynamic RNA‐protein machinery consisting of small nuclear ribonucleoproteins (snRNPs) and non‐snRNP proteins. Due to early evolutionary divergence, the amino acid sequences of trypanosome splicing factors exhibit limited similarity to those of their eukaryotic orthologs making their bioinformatic identification challenging. Most of the ～ 60 protein components that have been characterized thus far are snRNP proteins because, in contrast to individual snRNPs, purification of intact spliceosomes has not been achieved yet. Here, we characterize the non‐snRNP PRP19 complex of Trypanosoma brucei. We identified a complex that contained the core subunits PRP19, CDC5, PRL1, and SPF27, as well as PRP17, SKIP and PPIL1. Three of these proteins were newly annotated. The PRP19 complex was associated primarily with the activated spliceosome and, accordingly, SPF27 silencing blocked the first splicing step. Interestingly, SPF27 silencing caused an accumulation of SL RNA with a hypomethylated cap that closely resembled the defect observed previously upon depletion of the cyclin‐dependent kinase CRK9, indicating that both proteins may function in spliceosome activation.     

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis

Peptidyl-prolyl isomerase-like 1(PPIL1) is associated with the human spliceosome complex. However, its function in pre-mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18-2(AtCYP18-2), a PPIL1 homolog,plays an essential role in heat tolerance by regulating pre-mRNA splicing. Under heat stress conditions,AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP...     

Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development

BackgroundThe pervasive expression of circular RNA is a recently discovered feature of gene expression in highly diverged eukaryotes, but the functions of most circular RNAs are still unknown. Computational methods to discover and quantify circular RNA are essential. Moreover, discovering biological contexts where circular RNAs are regulated will shed light on potential functional roles they may play.ResultsWe present a new algorithm that increases the sensitivity and specificity of circular RNA detection by discovering and quantifying circular and linear RNA splicing events at both annotated and un-annotated exon boundaries, including intergenic regions of the genome, with high statistical confidence. Unlike approaches that rely on read count and exon homology to determine confidence in prediction of circular RNA expression, our algorithm uses a statistical approach. Using our algorithm, we unveiled striking induction of general and tissue-specific circular RNAs, including in the heart and lung, during human fetal development. We discover regions of the human fetal brain, such as the frontal cortex, with marked enrichment for genes where circular RNA isoforms are dominant.ConclusionsThe vast majority of circular RNA production occurs at major spliceosome splice sites; however, we find the first examples of developmentally induced circular RNAs processed by the minor spliceosome, and an enriched propensity of minor spliceosome donors to splice into circular RNA at un-annotated, rather than annotated, exons. Together, these results suggest a potentially significant role for circular RNA in human development.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0690-5) contains supplementary material, which is available to authorized users.     

Functions and regulation of the Brr2 RNA helicase during splicing

Pre-mRNA splicing entails the stepwise assembly of an inactive spliceosome, its catalytic activation, splicing catalysis and spliceosome disassembly. Transitions in this reaction cycle are accompanied by compositional and conformational rearrangements of the underlying RNA-protein interaction networks, which are driven and controlled by 8 conserved superfamily 2 RNA helicases. The Ski2-like helicase, Brr2, provides the key remodeling activity during spliceosome activation and is additionally implicated in the catalytic and disassembly phases of splicing, indicating that Brr2 needs to be tightly regulated during splicing. Recent structural and functional analyses have begun to unravel how Brr2 regulation is established via multiple layers of intra- and inter-molecular mechanisms. Brr2 has an unusual structure, including a long N-terminal region and a catalytically inactive C-terminal helicase cassette, which can auto-inhibit and auto-activate the enzyme, respectively. Both elements are essential, also serve as protein-protein interaction devices and the N-terminal region is required for stable Brr2 association with the tri-snRNP, tri-snRNP stability and retention of U5 and U6 snRNAs during spliceosome activation in vivo. Furthermore, a C-terminal region of the Prp8 protein, comprising consecutive RNase H-like and Jab1/MPN-like domains, can both up- and down-regulate Brr2 activity. Biochemical studies revealed an intricate cross-talk among the various cis- and trans-regulatory mechanisms. Comparison of isolated Brr2 to electron cryo-microscopic structures of yeast and human U4/U6?U5 tri-snRNPs and spliceosomes indicates how some of the regulatory elements exert their functions during splicing. The various modulatory mechanisms acting on Brr2 might be exploited to enhance splicing fidelity and to regulate alternative splicing.     

Plant-specific SR-related protein atSR45a interacts with spliceosomal proteins in plant nucleus

Serine/arginine-rich (SR) protein and its homologues (SR-related proteins) are important regulators of constitutive and/or alternative splicing and other aspects of mRNA metabolism. To clarify the contribution of a plant-specific and stress-responsive SR-related protein, atSR45a, to splicing events, here we analyzed the interaction of atSR45a with the other splicing factors by conducting a yeast two-hybrid assay and a bimolecular fluorescence complementation analysis. The atSR45a-1a and -2 proteins, the presumed mature forms produced by alternative splicing of atSR45a, interacted with U1-70K and U2AF³⁵b, splicing factors for the initial definition of 5′ and 3′ splice sites, respectively, in the early stage of spliceosome assembly. Both proteins also interacted with themselves, other SR proteins (atSR45 and atSCL28), and PRP38-like protein, a homologue of the splicing factor essential for cleavage of the 5′ splice site. The mapping of deletion mutants of atSR45a proteins revealed that the C-terminal arginine/serine-rich (RS) domain of atSR45a proteins are required for the interaction with U1-70K, U2AF³⁵b, atSR45, atSCL28, PRP38-like protein, and themselves, and the N-terminal RS domain enhances the interaction efficiency. Interestingly, the distinctive N-terminal extension in atSR45a-1a protein, but not atSR45a-2 protein, inhibited the interaction with these splicing factors. These findings suggest that the atSR45a proteins help to form the bridge between 5′ and 3′ splice sites in the spliceosome assembly and the efficiency of spliceosome formation is affected by the expression ratio of atSR45a-1a and atSR45a-2.