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The splicing of U12-type introns can be a rate-limiting step in gene expression.

ABSTRACT: Some protein-coding genes in metazoan genomes contain a minor class of introns that are excised by a distinct, low-abundance spliceosome. We have developed a quantitative RT-PCR assay that allows comparison of the relative rates of intron removal from the transcripts present in a pre-mRNA population. We show that the U12-type introns are more slowly spliced than the major-class (U2-type) introns from three endogenous pre-mRNAs in human tissue culture cells. In Drosophila melanogaster S2 cells, using minigene constructs designed to produce nearly identical mRNAs, we observe increased expression of fluorescent protein and mature mRNA upon mutation of a U12-type to a U2-type intron. These results provide evidence that the level of gene expression in vivo is lowered by the presence of a U12-type intron and implicate the U12-type spliceosome as a target in the post-transcriptional regulation of gene expression.

SUBMITTER: Patel AA

PROVIDER: S-EPMC126102 | biostudies-literature | 2002 Jul

REPOSITORIES: biostudies-literature

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The splicing of U12-type introns can be a rate-limiting step in gene expression.

Patel Abhijit A AA McCarthy Matthew M Steitz Joan A JA

The EMBO journal 20020701 14

Some protein-coding genes in metazoan genomes contain a minor class of introns that are excised by a distinct, low-abundance spliceosome. We have developed a quantitative RT-PCR assay that allows comparison of the relative rates of intron removal from the transcripts present in a pre-mRNA population. We show that the U12-type introns are more slowly spliced than the major-class (U2-type) introns from three endogenous pre-mRNAs in human tissue culture cells. In Drosophila melanogaster S2 cells, u ...[more]

PMID: 12110592

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

Project description:U12-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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Alternative splicing and bioinformatic analysis of human U12-type introns.

Project description:U12-type introns exist, albeit rarely, in a variety of multicellular organisms. Splicing of U12 intron-containing precursor mRNAs takes place in the U12-type spliceosome that is distinct from the major U2-type spliceosome. Due to incompatibility of these two spliceosomes, alternative splicing involving a U12-type intron may give rise to a relatively complicated impact on gene expression. We studied alternative U12-type intron splicing in an attempt to gain more mechanistic insights. First, we characterized mutually exclusive exon selection of the human JNK2 gene, which involves an unusual intron possessing the U12-type 5' splice site and the U2-type 3' splice site. We demonstrated that the long and evolutionary conserved polypyrimidine tract of this hybrid intron provides important signals for inclusion of its downstream alternative exon. In addition, we examined the effects of single nucleotide polymorphisms in the human WDFY1 U12-type intron on pre-mRNA splicing. These results provide mechanistic implications on splice-site selection of U12-type intron splicing. We finally discuss the potential effects of splicing of a U12-type intron with genetic defects or within a set of genes encoding RNA processing factors on global gene expression.

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U12-type spliceosomal introns of Insecta.

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Evolutionary dynamics of U12-type spliceosomal introns.

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U12 type introns were lost at multiple occasions during evolution.

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Branchpoint selection in the splicing of U12-dependent introns in vitro.

Project description:In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways. Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered. Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class. To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1 a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME). In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns. Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined. Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF. Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome.

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Extein residues play an intimate role in the rate-limiting step of protein trans-splicing.

Project description:Split inteins play an important role in modern protein semisynthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N- and C-exteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.

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Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome

Project description:Somatic mutations in the spliceosome gene ZRSR2 (located on the X chromosome) are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3' splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here, we characterize ZRSR2 as an essential component of the minor spliceosome (U12-dependent) assembly. shRNA mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns, and RNA-Sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns while splicing of the U2-type introns remain mostly unaffected. ZRSR2 deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS. RNA sequencing was performed on 16 bone marrow samples (MDS and normal) and six samples of control or ZRSR2 shRNA transduced TF-1 cells and data was analysed for aberrant splicing caused by ZRSR2 mutations/deficiency.

2015-01-15 | E-GEOD-63816 | biostudies-arrayexpress

Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome

Project description:Somatic mutations in the spliceosome gene ZRSR2— located on the X chromosome — are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3΄ splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here, we characterize ZRSR2 as an essential component of the minor spliceosome (U12-dependent) assembly. shRNA mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns, and RNA-Sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns while splicing of the U2-type introns remain mostly unaffected. ZRSR2 deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS.

2015-01-15 | GSE63816 | GEO

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