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Sequences of the 3 splice site mutants used in this study. The common 5 splice site is shown at the left. The 3 splice sites are shown at the right. The common branch site is shown in bold, and the mutated positions are underlined. The sites of in vivo splicing for each construct are indicated by the arrows. Major sites are indicated by dark arrowheads, and minor sites are indicated by light arrowheads. The numbers at the top refer to the distance in nucleotides between the branch site adenosine and the dashed lines.
Source publication
U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the
branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an
intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide...
Contexts in source publication
Context 1
... vitro splicing and spliceosome formation. DNA templates for in vitro transcription were prepared by PCR amplification using the minigene constructs with 3 splice site sequences shown in Fig. 2 as described (11). RNA was syn- thesized from these templates using T7 RNA polymerase and gel purified, and equal amounts of each RNA were spliced in vitro for 3 h in the presence of an antisense 2-O-methyl oligonucleotide directed against U2 snRNA as described (11). An antisense 2-O-methyl oligonucleotide against U12 snRNA was in- ...
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... con- structs were transfected into CHO cells, and RNA was har- vested after 48 h. The splicing pattern of the transfected P120 intron F was determined by reverse transcription (RT) of the RNA using a minigene-specific primer and PCR amplification of the products using primers in the flanking exons 6 and 7. Figure 2 shows the sequences of the mutants and indicates the sites of in vivo splicing used in each case. ...
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... 3 splice site in vivo, we varied this distance in the P120 intron between 8 and 27 nucleotides. The natural sequence around the wild-type AC 3 splice site was modified to create different spacings. We also generated additional mu- tations to determine the optimum spacing for this intron. The mutant constructs for these experiments are shown in Fig. 2. Figure 4 shows the RT-PCR results of in vivo splicing of P120 introns with a variety of branch site-to-3 splice site dis- tances. The wild-type P120 intron has a distance of 10 nucle- otides, the smallest distance seen in natural introns (see Fig. 1). As expected, all splicing was to the AC at the wild-type position (Fig. 4, lane 1). ...
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... of splicing also occurred at the 12 position, where there was a UU dinucleotide (location confirmed by sequencing). An AG dinucleotide positioned at 18 gave similar results (lane 4). To inactivate the 18 splice site, the A at 17 was mutated to C. In this mutant, P120 27 AC, the closest potential 3 splice sites become the AG at 25 and the AC at 27 (Fig. 2). The 25 AG is preceded by a consensus C residue, while the AC at 27 is preceded by a less 5). The solid line shows the distribution for all introns, while the black and grey bars show the distribution of distances in the AU-AC and GU-AG sub- classes of U12-dependent introns, respectively. The branch site is as- sumed to be the position ...
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... to determine the preferred distance be- tween the branch site and the 3 splice site. For this, we in- serted a sequence containing five repetitions of AC into the 3 splice site region so that potential 3 splice sites with the sequence CAC were available at distances of 10, 12, 14, 16, and 18 residues from the branch site adenosine (P120 oligo AC, Fig. 2). When this construct was assayed in vivo, splic- ing occurred almost exclusively at the 12 position (Fig. 4, lane 7). Note that in this construct, a completely wild- type CAC 3 splice site positioned 10 nucleotides from the branch site is ignored in favor of the site located 12 nucleo- tides from the branch site. A similar result was ...
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... lack of spliceosome formation activity of the 27 mu- tant was surprising, since Frilander and Steitz (14) have re- ported that an in vitro transcript which was truncated between the branch site and the 3 splice site was able to form both A and B complexes. We repeated this spliceosome complex result (P120 3 Trunc; Fig. 8, lanes 22 and 23) and also confirmed that this RNA was unable to carry out the first step of splicing to a detectable level (Fig. 6, lane 17). Thus, this truncated RNA, which is missing sequences downstream of the branch site, can still form spliceosome complexes, while the 27 AC mutant RNA, which has RNA but no functional splice sites down- stream of ...
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Citations
... mi-INTs key sequence features lie in the conservation of nucleotide segments in their 5 ss and BPS immediately upstream of their 3 ss [12]. The 5 ss conserved segment includes 8-9 nucleotides (nts) (A/G)UAUCCUUU, and the BPS includes the conserved sequence UCCUUAAC with fewer variations compared to U2 introns [12,19,44]. The splicing of mi-INTs depends on a well-situated 3 ss creating a functional constraint on the distance between the BPS and the 3 ss [44]. ...
... The 5 ss conserved segment includes 8-9 nucleotides (nts) (A/G)UAUCCUUU, and the BPS includes the conserved sequence UCCUUAAC with fewer variations compared to U2 introns [12,19,44]. The splicing of mi-INTs depends on a well-situated 3 ss creating a functional constraint on the distance between the BPS and the 3 ss [44]. Thus, this distance is relatively shorter (10-20 nts) in mi-INTs, with the optimal distance being 11-13 nts [44]. ...
... The splicing of mi-INTs depends on a well-situated 3 ss creating a functional constraint on the distance between the BPS and the 3 ss [44]. Thus, this distance is relatively shorter (10-20 nts) in mi-INTs, with the optimal distance being 11-13 nts [44]. Furthermore, mi-INTs lack a polypyrimidine tract which is commonly found in U2 introns ( Figure 1A). ...
Pre-mRNA splicing is an essential step in gene expression and is catalyzed by two machineries in eukaryotes: the major (U2 type) and minor (U12 type) spliceosomes. While the majority of introns in humans are U2 type, less than 0.4% are U12 type, also known as minor introns (mi-INTs), and require a specialized spliceosome composed of U11, U12, U4atac, U5, and U6atac snRNPs. The high evolutionary conservation and apparent splicing inefficiency of U12 introns have set them apart from their major counterparts and led to speculations on the purpose for their existence. However, recent studies challenged the simple concept of mi-INTs splicing inefficiency due to low abundance of their spliceosome and confirmed their regulatory role in alternative splicing, significantly impacting the expression of their host genes. Additionally, a growing list of minor spliceosome-associated diseases with tissue-specific pathologies affirmed the importance of minor splicing as a key regulatory pathway, which when deregulated could lead to tissue-specific pathologies due to specific alterations in the expression of some minor-intron-containing genes. Consequently, uncovering how mi-INTs splicing is regulated in a tissue-specific manner would allow for better understanding of disease pathogenesis and pave the way for novel therapies, which we highlight in this review.
... U12-type introns were first discovered due to their unusual AT-AC dinucleotide donor and acceptor splice sites and believed to harbor exclusively these sequences (Jackson 1991). They are now computationally identified based on their specific donor splice site and branch point sequence (BPS) consensus sequences, the latter being located within a specific window of 10-13 nt before the acceptor splice site (Dietrich et al. 2001a(Dietrich et al. , 2005. In 2007, these criteria enabled to identify 695 introns of the U12-type in the human genome, thus representing <1% of all human introns (Alioto 2007). ...
Minor intron splicing plays a central role in human embryonic development and survival. Indeed, biallelic mutations in RNU4ATAC, transcribed into the minor spliceosomal U4atac snRNA, are responsible for three rare autosomal recessive multimalformation disorders named Taybi-Linder (TALS/MOPD1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes, which associate numerous overlapping signs of varying severity. Although RNA-seq experiments have been conducted on a few RFMN patient cells, none have been performed in TALS, and more generally no in-depth transcriptomic analysis of the ∼700 human genes containing a minor (U12-type) intron had been published as yet. We thus sequenced RNA from cells derived from five skin, three amniotic fluid, and one blood biosamples obtained from seven unrelated TALS cases and from age- and sex-matched controls. This allowed us to describe for the first time the mRNA expression and splicing profile of genes containing U12-type introns, in the context of a functional minor spliceosome. Concerning RNU4ATAC-mutated patients, we show that as expected, they display distinct U12-type intron splicing profiles compared to controls, but that rather unexpectedly mRNA expression levels are mostly unchanged. Furthermore, although U12-type intron missplicing concerns most of the expressed U12 genes, the level of U12-type intron retention is surprisingly low in fibroblasts and amniocytes, and much more pronounced in blood cells. Interestingly, we found several occurrences of introns that can be spliced using either U2, U12, or a combination of both types of splice site consensus sequences, with a shift towards splicing using preferentially U2 sites in TALS patients' cells compared to controls.
... Here, we used a stretch of 12 nucleotides for the 5′SS (− 3 to + 9), and 14 nucleotides for the 3′SS (− 13 to + 1). The branch point sequence (BPS) of minor introns has been shown to be closely positioned to the 3′SS, with an optimal distance of 11-13 nucleotides [45]. As such, we curated a sequence up to 40 nucleotides upstream of the 3′SS (− 40 to − 1) and then isolated 29 windows of 12 nucleotides in length, thus resulting in 29 putative BPS. ...
Background:
Mutations in minor spliceosome components such as U12 snRNA (cerebellar ataxia) and U4atac snRNA (microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1)) result in tissue-specific symptoms. Given that the minor spliceosome is ubiquitously expressed, we hypothesized that these restricted phenotypes might be caused by the tissue-specific regulation of the minor spliceosome targets, i.e. minor intron-containing genes (MIGs). The current model of inefficient splicing is thought to apply to the regulation of the ~ 500 MIGs identified in the U12DB. However this database was created more than 10 years ago. Therefore, we first wanted to revisit the classification of minor introns in light of the most recent reference genome. We then sought to address specificity of MIG expression, minor intron retention, and alternative splicing (AS) across mouse and human tissues.
Results:
We employed position-weight matrices to obtain a comprehensive updated list of minor introns, consisting of 722 mouse and 770 human minor introns. These can be found in the Minor Intron DataBase (MIDB). Besides identification of 99% of the minor introns found in the U12DB, we also discovered ~ 150 new MIGs. We then analyzed the RNAseq data from eleven different mouse tissues, which revealed tissue-specific MIG expression and minor intron retention. Additionally, many minor introns were efficiently spliced compared to their flanking major introns. Finally, we identified several novel AS events across minor introns in both mouse and human, which were also tissue-dependent. Bioinformatics analysis revealed that several of the AS events could result in the production of novel tissue-specific proteins. Moreover, like the major introns, we found that these AS events were more prevalent in long minor introns, while retention was favoured in shorter introns.
Conclusion:
Here we show that minor intron splicing and AS across minor introns is a highly organised process that might be regulated in coordination with the major spliceosome in a tissue-specific manner. We have provided a framework to further study the impact of the minor spliceosome and the regulation of MIG expression. These findings may shed light on the mechanism underlying tissue-specific phenotypes in diseases associated with minor spliceosome inactivation. MIDB can be accessed at https://midb.pnb.uconn.edu .
... Branchpoints within U12-type introns exhibited a bimodal distribution (Fig. 2B). Approximately half of such U12-type branchpoints were found in close proximity (within 20 nt) of the 3 ′ splice site, as observed previously (Dietrich et al. 2001;Taggart et al. 2017). In contrast, approximately half of U12-type introns were located only modestly closer to the 3 ′ splice site than we observed for U2-type branchpoints. ...
Although branchpoint recognition is an essential component of intron excision during the RNA splicing process, the branchpoint itself is frequently assumed to be a basal, rather than regulatory, sequence feature. However, this assumption has not been systematically tested due to the technical difficulty of identifying branchpoints and quantifying their usage. Here, we analyzed ∼1.31 trillion reads from 17,164 RNA sequencing data sets to demonstrate that almost all human introns contain multiple branchpoints. This complexity holds even for constitutive introns, 95% of which contain multiple branchpoints, with an estimated five to six branchpoints per intron. Introns upstream of the highly regulated ultraconserved poison exons of SR genes contain twice as many branchpoints as the genomic average. Approximately three-quarters of constitutive introns exhibit tissue-specific branchpoint usage. In an extreme example, we observed a complete switch in branchpoint usage in the well-studied first intron of HBB (β-globin) in normal bone marrow versus metastatic prostate cancer samples. Our results indicate that the recognition of most introns is unexpectedly complex and tissue-specific and suggest that alternative splicing catalysis typifies the majority of introns even in the absence of differences in the mature mRNA.
... Several extremely rare terminal intron sequences were discovered and often discussed as potential artifacts, e.g. introns with GT-GG or TT-AG termini [11][12][13][14]. Further details regarding exceptional splicing events have recently been reviewed [15,16]. ...
Objective: The Arabidopsis thaliana Niederzenz-1 genome sequence was recently published with an ab initio gene
prediction. In depth analysis of the predicted gene set revealed some errors involving genes with non-canonical
splice sites in their introns. Since non-canonical splice sites are difficult to predict ab initio, we checked for options
to improve the annotation by transferring annotation information from the recently released Columbia-0 reference
genome sequence annotation Araport11.
Results: Incorporation of hints generated from Araport11 enabled the precise prediction of non-canonical splice
sites. Manual inspection of RNA-Seq read mapping and RT-PCR were applied to validate the structural annotations
of non-canonical splice sites. Predictions of untranslated regions were also updated by harnessing the potential of
Araport11’s information, which was generated by using high coverage RNA-Seq data. The improved gene set of the
Nd-1 genome assembly (GeneSet_Nd-1_v1.1) was evaluated via comparison to the initial gene prediction (Gen-
eSet_Nd-1_v1.0) as well as against Araport11 for the Col-0 reference genome sequence. GeneSet_Nd-1_v1.1 contains
previously missed non-canonical splice sites in 1256 genes. Reciprocal best hits for 24,527 (89.4%) of all nuclear Col-0
genes against the GeneSet_Nd-1_v1.1 indicate a high gene prediction quality.
... The branchpoint distribution in yeast (Schizosaccharomyces pombe) was shifted significantly closer to the 3 ′ ss than their mammalian counterparts. Similarly, 255 branchpoints that correspond to U12 minor introns were positioned relatively closer to the 3 ′ ss (n = 255, P-value < 0.001) as reported previously (Supplemental Fig. S2; Mertins and Gallwitz 1987;Dietrich et al. 2001). Approximately 9% of human U2 branchpoints were found <10 nt from the 3 ′ ss AG. ...
The coding sequence of each human pre-mRNA is interrupted, on average, by eleven introns that must be spliced out for proper gene expression. Each intron contains three obligate signals: a 5' splice site, a branch site and a 3' splice site. Splice site usage has been mapped exhaustively across different species, cell types and cellular states. In contrast, only a small fraction of branch sites have been identified even once. The few reported annotations of branch site are imprecise as reverse transcriptase skips several nucleotides while traversing a 2-5 linkage. Here, we report large-scale mapping of the branchpoints from deep sequencing data in three different species and in the SF3B1 K700E oncogenic mutant background. We have developed a novel method whereby raw lariat reads are refined by U2snRNP/pre-mRNA basepairing models to return the largest current dataset of branchpoint sequences with quality metrics. This analysis discovers novel modes of U2snRNA:pre-mRNA basepairing conserved in yeast and provides insight into the biogenesis of intron circles. Finally, matching branchsite usage with isoform selection across the extensive panel of ENCODE RNA-seq datasets, offers insight into the mechanisms by which branchpoint usage drives alternative splicing.
... Besides, the thermodynamic stability of this interaction 104 is vital for proper splicing of U12-type pre-mRNAs in the absence of a polypyrimidine (Py) tract and increased distance between the degenerate 3 0 splice sites from the branch point. 105 Therefore, it can be speculated that the proteins SF3b49, SF3b145 as well as p14 act as "molecular rulers" to improve the fidelity of branch point adenosine selection both by stabilizing and proof-reading the U12 snRNA: pre-mRNA interaction (Fig. 7). We compared the centroid positions of these proteins in both the SF3b closed form and SF3b open form-RNA complex and observed changes both in terms of orientation and distances (Fig. 7). ...
Pre-mRNA splicing in eukaryotes is performed by the spliceosome, a highly complex macromolecular machine. SF3b is a multi-protein complex which recognizes the branch point adenosine of pre-mRNA as part of a larger U2 snRNP or U11/U12 di-snRNP in the dynamic spliceosome machinery. Although a cryo-EM map is available for human SF3b complex, the structure and relative spatial arrangement of all components in the complex are not yet known. We have recognized folds of domains in various proteins in the assembly and generated comparative models. Using an integrative approach involving structural and other experimental data, guided by the available cryo-EM density map, we deciphered a pseudo-atomic model of the closed form of SF3b which is found to be a "fuzzy complex" with highly flexible components and multiplicity of folds. Further, the model provides structural information for 5 proteins (SF3b10, SF3b155, SF3b145, SF3b130 and SF3b14b) and localization information for 4 proteins (SF3b10, SF3b145, SF3b130 and SF3b14b) in the assembly for the first time. Integration of this model with the available U11/U12 di-snRNP cryo-EM map enabled elucidation of an open form. This now provides new insights on the mechanistic features involved in the transition between closed and open forms pivoted by a hinge region in the SF3b155 protein that also harbors cancer causing mutations. Moreover, the open form guided model of the 5' end of U12 snRNA, which includes the branch point duplex, shows that the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation with potential implications for branch point adenosine recognition fidelity.
... Typically, U2 introns have GU dinucleotide at the 5'-splice site, AG dinucleotide at the 3' -splice site, and CURACU sequence at the branch site, where the A is the branch point adenosine. There is also a pyrimidine rich region between the branch and 3' splice sites in U2-dependent introns [Dietrich et al., 2001]. ...
Splicing is a highly regulated process which plays a significant role in eukaryotic genes expression, and is vital for cell function. Understanding splicing mechanisms and core components involved in this process will allow defining causes of various diseases. Splicing is performed by complex ribonucleoprotein particles named spliceosomes. Mammals have two types of spliceosomes (termed the minor and the major spliceosomes), each one comprised of five snRNPs and numerous proteins. Spliceosomes differ in their compartments, recognition sites and recognition processes, but they have similar structure. Although spliceosomes’ functions are equivalent, they are not permutable, so defects in the major or minor one lead to various diseases. Recent studies have shown that mutations in splicing-associated genes linked with such disorders as spinal muscular atrophy, autosomal dominant retinitis pigmentosum, microcephalic osteodysplastic primordial dwarfism type I. In addition, very exciting recent results of RNA deep-sequencing demonstrate splicing abnormalities in Alzheimer’s disease.
... branch point site (BPS) TCCTTAACT and A(C/G) at 3 " splice site [8,111213. However, as reported recently by Lin et al. ...
... U12-type introns can be flanked by different terminal dinucleotides indicating that the donor and acceptor sites are degenerate [14]. The BPS of U2-type introns is usually located 18-40 nucleotides upstream of the 3 " splice site, in contrast to the U12-type introns, where it is restricted to 12-15 nu- cleotides [8,1112 15]. Additionally, U12-type introns lack a polypyrimidine tract between the BPS and the 3′ splice site. ...
Most of eukaryotic genes are interrupted by introns that need to be removed from pre-mRNAs before they can perform their function. This is done by complex machinery called spliceosome. Many eukaryotes possess two separate spliceosomal systems that process separate sets of introns. The major (U2) spliceosome removes majority of introns, while minute fraction of intron repertoire is processed by the minor (U12) spliceosome. These two populations of introns are called U2-type and U12-type, respectively. The latter fall into two subtypes based on the terminal dinucleotides. The minor spliceosomal system has been lost independently in some lineages, while in some others few U12-type introns persist. We investigated twenty insect genomes in order to better understand the evolutionary dynamics of U12-type introns. Our work confirms dramatic drop of U12-type introns in Diptera, leaving these genomes just with a handful cases. This is mostly the result of intron deletion, but in a number of dipteral cases, minor type introns were switched to a major type, as well. Insect genes that harbor U12-type introns belong to several functional categories among which proteins binding ions and nucleic acids are enriched and these few categories are also overrepresented among these genes that preserved minor type introns in Diptera.
... As discussed above, splicing of U11 and U12-type introns requires U11 and U12 snRNAs.Figure 2D shows that, following oligonucleotide-directed RNase H degradation of either U11 or U12 snRNA, the Urp–P120 39 splice site interaction did not occur. Previous studies have shown that mutating the C of the 39 splice site AC dinucleotide does not impair splicing of a U12-type intron, whereas mutation of the A is detrimental (Dietrich et al. 2001Dietrich et al. , 2005). To determine whether these splicing results correlated with binding of Urp to the 39 splice site, we analyzed a series of P120 derivatives that have been characterized previously for splicing activity (Dietrich et al. 2001Dietrich et al. , 2005). ...
... Previous studies have shown that mutating the C of the 39 splice site AC dinucleotide does not impair splicing of a U12-type intron, whereas mutation of the A is detrimental (Dietrich et al. 2001Dietrich et al. , 2005). To determine whether these splicing results correlated with binding of Urp to the 39 splice site, we analyzed a series of P120 derivatives that have been characterized previously for splicing activity (Dietrich et al. 2001Dietrich et al. , 2005). The UV-cross-linking experiment ofFigure 2E shows that mutation of the 39 splice site AC dinucleotide to either AU, AA, or AG had no effect on binding of Urp. ...
The U2AF35-related protein Urp has been implicated previously in splicing of the major class of U2-type introns. Here we show that Urp is also required for splicing of the minor class of U12-type introns. Urp is recruited in an ATP-dependent fashion to the U12-type intron 3' splice site, where it promotes formation of spliceosomal complexes. Remarkably, Urp also contacts the 3' splice site of a U2-type intron, but in this case is specifically required for the second step of splicing. Thus, through recognition of a common splicing element, Urp facilitates distinct steps of U2- and U12-type intron splicing.
