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1.
Nat Commun. 2018 Aug 17;9(1):3315. doi: 10.1038/s41467-018-05748-7.

Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis.

Author information

1
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.
2
Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438, Frankfurt, Germany.
3
Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028, Lisboa, Portugal.
4
Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, Campus Gambelas, 8005-139, Faro, Portugal.
5
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. s.legewie@imb-mainz.de.
6
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. j.koenig@imb-mainz.de.
7
Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438, Frankfurt, Germany. kathi.zarnack@bmls.de.

Abstract

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.

2.
Nucleic Acids Res. 2018 Jul 2;46(W1):W25-W29. doi: 10.1093/nar/gky329.

Freiburg RNA tools: a central online resource for RNA-focused research and teaching.

Author information

1
Bioinformatics, Computer Science, University of Freiburg, Georges-Koehler-Allee 106, 79110 Freiburg, Germany.
2
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.
3
Department of Computer Science, University of Exeter, Exeter EX4 4QF, UK.
4
Coreva Scientific, Kaiser-Joseph-Str 198-200, 79098 Freiburg, Germany.
5
Genetics and Experimental Bioinformatics, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany.
6
Max Planck Institute of Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg, Germany.
7
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
8
Max Planck Institute for the Physics of Complex Systems, Dresden, Germany.
9
Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Long Road, Cambridge CB2 0PT, UK.
10
Department of Human Genetics, The Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1HH, UK.
11
Genedata AG, Margarethenstrasse 38, 4053 Basel, Switzerland.
12
Theoretical Biochemistry Group, University of Vienna, Währingerstraße 17, 1090 Vienna, Austria.
13
Department of Clinical Research, Clinical Trial Unit, University of Basel Hospital, Schanzenstrasse 55, 4031 Basel, Switzerland.
14
Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Schaenzlestr. 18, 79104 Freiburg, Germany.

Abstract

The Freiburg RNA tools webserver is a well established online resource for RNA-focused research. It provides a unified user interface and comprehensive result visualization for efficient command line tools. The webserver includes RNA-RNA interaction prediction (IntaRNA, CopraRNA, metaMIR), sRNA homology search (GLASSgo), sequence-structure alignments (LocARNA, MARNA, CARNA, ExpaRNA), CRISPR repeat classification (CRISPRmap), sequence design (antaRNA, INFO-RNA, SECISDesign), structure aberration evaluation of point mutations (RaSE), and RNA/protein-family models visualization (CMV), and other methods. Open education resources offer interactive visualizations of RNA structure and RNA-RNA interaction prediction as well as basic and advanced sequence alignment algorithms. The services are freely available at http://rna.informatik.uni-freiburg.de.

3.
Genome Res. 2018 May;28(5):699-713. doi: 10.1101/gr.229757.117. Epub 2018 Apr 11.

In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors.

Author information

1
Institute of Molecular Biology (IMB) gGmbH, 55128 Mainz, Germany.
2
Institute of Structural Biology, Helmholtz Center Munich, 85764 Neuherberg, Germany.
3
Biomolecular NMR and Center for Integrated Protein Science Munich at Department of Chemistry, Technical University of Munich, 85747 Garching, Germany.
4
Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, University of Leipzig, 04107 Leipzig, Germany.
5
Bioinformatics Group, Department of Computer Science, University of Freiburg, 79110 Freiburg, Germany.
6
Centre for Biological Signalling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany.
7
Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, 60438 Frankfurt a.M., Germany.
#
Contributed equally

Abstract

Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes. The RNA-binding protein (RBP) U2AF2 is central to splicing decisions, as it recognizes 3' splice sites and recruits the spliceosome. We establish "in vitro iCLIP" experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF2 recognizes RNA sequences and how this is modulated by trans-acting RBPs. We measure U2AF2 affinities at hundreds of binding sites and compare in vitro and in vivo binding landscapes by mathematical modeling. We find that trans-acting RBPs extensively regulate U2AF2 binding in vivo, including enhanced recruitment to 3' splice sites and clearance of introns. Using machine learning, we identify and experimentally validate novel trans-acting RBPs (including FUBP1, CELF6, and PCBP1) that modulate U2AF2 binding and affect splicing outcomes. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation.

PMID:
29643205
PMCID:
PMC5932610
DOI:
10.1101/gr.229757.117
[Indexed for MEDLINE]
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4.
RNA Biol. 2016 Jul 2;13(7):646-55. doi: 10.1080/15476286.2016.1191727. Epub 2016 May 31.

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns.

Author information

1
a Department of Microbiology and Molecular Genetics , School of Medicine, University of California , Irvine , CA , USA.
2
b Institute of Molecular Biology (IMB) , Mainz , Germany.

Abstract

Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.

KEYWORDS:

Alternative polyadenylation; CFIm25; CstF64; HnRNP A2/B1; PAS-Seq; RNA-Seq; alternative splicing

PMID:
27245359
PMCID:
PMC4962795
DOI:
10.1080/15476286.2016.1191727
[Indexed for MEDLINE]
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5.
PLoS Genet. 2015 Mar 25;11(3):e1005097. doi: 10.1371/journal.pgen.1005097. eCollection 2015 Mar.

Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

Author information

1
Department of Developmental and Cellular Biology, School of Biological Sciences, University of California, Irvine, Irvine, California, United States of America; CHOC Childrens', Division of Metabolics, Orange, California, United States of America.
2
Department of Otorhinolaryngology Head & Neck Surgery, School of Medicine, University of Maryland, Baltimore, Maryland, United States of America.
3
Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
4
National Center for Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
5
Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
6
Division of Pediatric Otolaryngology Head & Neck Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
7
Smilow Center for Translational Research, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
8
Trovagene, San Diego, California, United States of America.
9
Marshall B Ketchum University, Fullerton, California, United States of America.
10
Department of Physiology and Biophysics, University of California, Irvine, Irvine, California, United States of America.
11
CHOC Childrens', Division of Metabolics, Orange, California, United States of America.
12
Manchester Centre for Genomic Medicine, University of Manchester and Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre (MAHSC), Manchester, United Kingdom.
13
Department of Neurology and Neurobiology, University of California, Irvine, Irvine, California, United States of America.
14
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
15
Swiss Epilepsy Center, Zurich, Switzerland.
16
Institute of Molecular Biology, Mainz, Germany.
17
Department of Developmental and Cellular Biology, School of Biological Sciences, University of California, Irvine, Irvine, California, United States of America.
18
Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California, United States of America.
19
Architecture et Réactivité de l'ARN, CNRS, University of Strasbourg, IBMC, Strasbourg, France.
20
Parkinson's Institute and Clinical Center, Sunnyvale, California, United States of America.
21
Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, United States of America.
22
Section on Sensory Cell Regeneration and Development, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, United States of America.
23
Biochemistry and Genetics Department, UMR CNRS 6214-INSERM U1083, CHU Angers, Angers, France.
24
Jinnah Hospital Complex, Allama Iqbal Medical College, University of Health Sciences, Lahore, Pakistan; University of Lahore, Lahore, Pakistan; Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan.

Abstract

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

PMID:
25807530
PMCID:
PMC4373692
DOI:
10.1371/journal.pgen.1005097
[Indexed for MEDLINE]
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6.
RNA. 2015 May;21(5):813-23. doi: 10.1261/rna.048769.114. Epub 2015 Mar 24.

Splicing predictions reliably classify different types of alternative splicing.

Author information

1
Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697-4025, USA Institute of Molecular Biology (IMB), D-55128 Mainz, Germany.
2
Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697-4025, USA.

Abstract

Alternative splicing is a key player in the creation of complex mammalian transcriptomes and its misregulation is associated with many human diseases. Multiple mRNA isoforms are generated from most human genes, a process mediated by the interplay of various RNA signature elements and trans-acting factors that guide spliceosomal assembly and intron removal. Here, we introduce a splicing predictor that evaluates hundreds of RNA features simultaneously to successfully differentiate between exons that are constitutively spliced, exons that undergo alternative 5' or 3' splice-site selection, and alternative cassette-type exons. Surprisingly, the splicing predictor did not feature strong discriminatory contributions from binding sites for known splicing regulators. Rather, the ability of an exon to be involved in one or multiple types of alternative splicing is dictated by its immediate sequence context, mainly driven by the identity of the exon's splice sites, the conservation around them, and its exon/intron architecture. Thus, the splicing behavior of human exons can be reliably predicted based on basic RNA sequence elements.

KEYWORDS:

alternative splicing; bioinformatics; splicing predictor; support vector machine

PMID:
25805853
PMCID:
PMC4408789
DOI:
10.1261/rna.048769.114
[Indexed for MEDLINE]
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7.
Nucleic Acids Res. 2014 Dec 16;42(22):13615-32. doi: 10.1093/nar/gku1186. Epub 2014 Nov 20.

The TCF C-clamp DNA binding domain expands the Wnt transcriptome via alternative target recognition.

Author information

1
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697, USA.
2
Department of Information and Computer Science, University of California, Irvine, Irvine, CA 92697, USA.
3
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697, USA marian.waterman@uci.edu.

Abstract

LEF/TCFs direct the final step in Wnt/β-catenin signalling by recruiting β-catenin to genes for activation of transcription. Ancient, non-vertebrate TCFs contain two DNA binding domains, a High Mobility Group box for recognition of the Wnt Response Element (WRE; 5'-CTTTGWWS-3') and the C-clamp domain for recognition of the GC-rich Helper motif (5'-RCCGCC-3'). Two vertebrate TCFs (TCF-1/TCF7 and TCF-4/TCF7L2) use the C-clamp as an alternatively spliced domain to regulate cell-cycle progression, but how the C-clamp influences TCF binding and activity genome-wide is not known. Here, we used a doxycycline inducible system with ChIP-seq to assess how the C-clamp influences human TCF1 binding genome-wide. Metabolic pulse-labeling of nascent RNA with 4'Thiouridine was used with RNA-seq to connect binding to the Wnt transcriptome. We find that the C-clamp enables targeting to a greater number of gene loci for stronger occupancy and transcription regulation. The C-clamp uses Helper sites concurrently with WREs for gene targeting, but it also targets TCF1 to sites that do not have readily identifiable canonical WREs. The coupled ChIP-seq/4'Thiouridine-seq analysis identified new Wnt target genes, including additional regulators of cell proliferation. Thus, C-clamp containing isoforms of TCFs are potent transcriptional regulators with an expanded transcriptome directed by C-clamp-Helper site interactions.

PMID:
25414359
PMCID:
PMC4267635
DOI:
10.1093/nar/gku1186
[Indexed for MEDLINE]
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8.
PLoS One. 2014 May 1;9(5):e95486. doi: 10.1371/journal.pone.0095486. eCollection 2014.

Dicer regulates differentiation and viability during mouse pancreatic cancer initiation.

Author information

1
Diabetes Center, Department of Medicine, University of California San Francisco, San Francisco, California, United States of America.
2
Department of Pathology, University of California San Francisco, San Francisco, California, United States of America.
3
Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California, United States of America.
4
Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, United States of America.

Abstract

miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.

PMID:
24788257
PMCID:
PMC4006805
DOI:
10.1371/journal.pone.0095486
[Indexed for MEDLINE]
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9.
Nat Cell Biol. 2014 Mar;16(3):255-67. doi: 10.1038/ncb2916. Epub 2014 Feb 23.

The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma.

Author information

1
1] Diabetes Center, Department of Medicine, University of California, San Francisco, 513 Parnassus Avenue, HSW 1116, Box 0540 San Francisco, California 94143, USA [2] II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany [3].
2
1] Diabetes Center, Department of Medicine, University of California, San Francisco, 513 Parnassus Avenue, HSW 1116, Box 0540 San Francisco, California 94143, USA [2] Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan [3].
3
1] Diabetes Center, Department of Medicine, University of California, San Francisco, 513 Parnassus Avenue, HSW 1116, Box 0540 San Francisco, California 94143, USA [2].
4
Diabetes Center, Department of Medicine, University of California, San Francisco, 513 Parnassus Avenue, HSW 1116, Box 0540 San Francisco, California 94143, USA.
5
Department of Pathology, University of California, San Francisco, San Francisco, California 94143, USA.
6
1] Department of Surgery [2] Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84115, USA.
7
Department of Pathology, University of California, Los Angeles, California 90095, USA.
8
Department of Medicine, Imperial College London, W12 ONN London, UK.
9
Department of Microbiology & Molecular Genetics, University of California, Irvine, California 92697, USA.

Abstract

Pancreatic ductal adenocarcinoma (PDA) develops through distinct precursor lesions, including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). However, genetic features resulting in IPMN-associated PDA (IPMN-PDA) versus PanIN-associated PDA (PanIN-PDA) are largely unknown. Here we find that loss of Brg1, a core subunit of SWI/SNF chromatin remodelling complexes, cooperates with oncogenic Kras to form cystic neoplastic lesions that resemble human IPMN and progress to PDA. Although Brg1-null IPMN-PDA develops rapidly, it possesses a distinct transcriptional profile compared with PanIN-PDA driven by mutant Kras and hemizygous p53 deletion. IPMN-PDA also is less lethal, mirroring prognostic trends in PDA patients. In addition, Brg1 deletion inhibits Kras-dependent PanIN development from adult acinar cells, but promotes Kras-driven preneoplastic transformation in adult duct cells. Therefore, this study implicates Brg1 as a determinant of context-dependent Kras-driven pancreatic tumorigenesis and suggests that chromatin remodelling may underlie the development of distinct PDA subsets.

Comment in

PMID:
24561622
PMCID:
PMC4684081
DOI:
10.1038/ncb2916
[Indexed for MEDLINE]
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10.
J Med Genet. 2014 Mar;51(3):185-96. doi: 10.1136/jmedgenet-2013-101660. Epub 2014 Jan 15.

A splice donor mutation in NAA10 results in the dysregulation of the retinoic acid signalling pathway and causes Lenz microphthalmia syndrome.

Author information

1
Department of Pediatrics, Division of Human Genetics, University of California Irvine, Irvine, California, USA.

Abstract

INTRODUCTION:

Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition.

METHODS AND RESULTS:

Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells.

CONCLUSIONS:

We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway.

KEYWORDS:

Clinical Genetics; Developmental; Genome-Wide; Lenz Microphthalmia Syndrome; NAA10

PMID:
24431331
PMCID:
PMC4278941
DOI:
10.1136/jmedgenet-2013-101660
[Indexed for MEDLINE]
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11.
RNA. 2013 Jan;19(1):96-102. doi: 10.1261/rna.037044.112. Epub 2012 Nov 21.

Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms.

Author information

1
Institute of Virology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany.

Erratum in

  • RNA. 2013 Jul;19(7):1015.

Abstract

Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5' splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection.

PMID:
23175589
PMCID:
PMC3527730
DOI:
10.1261/rna.037044.112
[Indexed for MEDLINE]
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12.
Nucleic Acids Res. 2013 Jan;41(Database issue):D118-24. doi: 10.1093/nar/gks969. Epub 2012 Oct 31.

HEXEvent: a database of Human EXon splicing Events.

Author information

1
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697-4025, USA.

Abstract

HEXEvent (http://hexevent.mmg.uci.edu) is a new database that permits the user to compile genome-wide exon data sets of human internal exons showing selected splicing events. User queries can be customized based on the type and the frequency of alternative splicing events. For each splicing version of an exon, an ESTs count is given, specifying the frequency of the event. A user-specific definition of constitutive exons can be entered to designate an exon exclusion level still acceptable for an exon to be considered as constitutive. Similarly, the user has the option to define a maximum inclusion level for an exon to be called an alternatively spliced exon. Unlike other existing splicing databases, HEXEvent permits the user to easily extract alternative splicing information for individual, multiple or genome-wide human internal exons. Importantly, the generated data sets are downloadable for further analysis.

PMID:
23118488
PMCID:
PMC3531206
DOI:
10.1093/nar/gks969
[Indexed for MEDLINE]
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13.
Genome Biol. 2012 Feb 22;13(2):143. doi: 10.1186/gb-2012-13-2-143.

Extensive regulation of NAGNAG alternative splicing: new tricks for the spliceosome?

Author information

1
Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, Irvine, CA 92697-4025, USA.

Abstract

A recent study using massive parallel sequencing demonstrates unequivocally that alternative tandem acceptor splicing is tissue-specifically regulated.

PMID:
22356731
PMCID:
PMC3334559
DOI:
10.1186/gb-2012-13-2-143
[Indexed for MEDLINE]
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14.
Wiley Interdiscip Rev RNA. 2012 Jan-Feb;3(1):1-12. doi: 10.1002/wrna.100. Epub 2011 Sep 2.

Evolution of SR protein and hnRNP splicing regulatory factors.

Author information

1
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA.

Abstract

The splicing of pre-mRNAs is an essential step of gene expression in eukaryotes. Introns are removed from split genes through the activities of the spliceosome, a large ribonuclear machine that is conserved throughout the eukaryotic lineage. While unicellular eukaryotes are characterized by less complex splicing, pre-mRNA splicing of multicellular organisms is often associated with extensive alternative splicing that significantly enriches their proteome. The alternative selection of splice sites and exons permits multicellular organisms to modulate gene expression patterns in a cell type-specific fashion, thus contributing to their functional diversification. Alternative splicing is a regulated process that is mainly influenced by the activities of splicing regulators, such as SR proteins or hnRNPs. These modular factors have evolved from a common ancestor through gene duplication events to a diverse group of splicing regulators that mediate exon recognition through their sequence-specific binding to pre-mRNAs. Given the strong correlations between intron expansion, the complexity of pre-mRNA splicing, and the emergence of splicing regulators, it is argued that the increased presence of SR and hnRNP proteins promoted the evolution of alternative splicing through relaxation of the sequence requirements of splice junctions.

PMID:
21898828
PMCID:
PMC3235224
DOI:
10.1002/wrna.100
[Indexed for MEDLINE]
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15.
Nucleic Acids Res. 2011 Nov 1;39(20):8928-37. doi: 10.1093/nar/gkr481. Epub 2011 Jul 27.

Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites.

Author information

1
Department of Microbiology & Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA.

Abstract

Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. In vertebrates, most splice sites are initially recognized by the spliceosome across the exon, because most exons are small and surrounded by large introns. This gene architecture predicts that efficient exon recognition depends largely on the strength of the flanking 3' and 5' splice sites. However, it is unknown if the 3' or the 5' splice site dominates the exon recognition process. Here, we test the 3' and 5' splice site contributions towards efficient exon recognition by systematically replacing the splice sites of an internal exon with sequences of different splice site strengths. We show that the presence of an optimal splice site does not guarantee exon inclusion and that the best predictor for exon recognition is the sum of both splice site scores. Using a genome-wide approach, we demonstrate that the combined 3' and 5' splice site strengths of internal exons provide a much more significant separator between constitutive and alternative exons than either the 3' or the 5' splice site strength alone.

PMID:
21795381
PMCID:
PMC3203598
DOI:
10.1093/nar/gkr481
[Indexed for MEDLINE]
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16.
Bioinformatics. 2008 Dec 15;24(24):2849-56. doi: 10.1093/bioinformatics/btn544. Epub 2008 Oct 21.

IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions.

Author information

1
Bioinformatics Group, Albert-Ludwigs-University Freiburg, Georges-Koehler-Allee 106, Freiburg D-79110, Germany. IntaRNA@informatik.uni-freiburg.de

Abstract

MOTIVATION:

During the last few years, several new small regulatory RNAs (sRNAs) have been discovered in bacteria. Most of them act as post-transcriptional regulators by base pairing to a target mRNA, causing translational repression or activation, or mRNA degradation. Numerous sRNAs have already been identified, but the number of experimentally verified targets is considerably lower. Consequently, computational target prediction is in great demand. Many existing target prediction programs neglect the accessibility of target sites and the existence of a seed, while other approaches are either specialized to certain types of RNAs or too slow for genome-wide searches.

RESULTS:

We introduce INTARNA, a new general and fast approach to the prediction of RNA-RNA interactions incorporating accessibility of target sites as well as the existence of a user-definable seed. We successfully applied INTARNA to the prediction of bacterial sRNA targets and determined the exact locations of the interactions with a higher accuracy than competing programs.

AVAILABILITY:

http://www.bioinf.uni-freiburg.de/Software/

PMID:
18940824
PMCID:
PMC2639303
DOI:
10.1093/bioinformatics/btn544
[Indexed for MEDLINE]
Free PMC Article
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17.
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W310-3. Epub 2007 Apr 22.

INFO-RNA--a server for fast inverse RNA folding satisfying sequence constraints.

Author information

1
Albert-Ludwigs-University Freiburg, Institute of Computer Science, Bioinformatics Group, Georges-Koehler-Allee 106, 79110 Freiburg, Germany. abusch@informatik.uni-freiburg.de

Abstract

INFO-RNA is a new web server for designing RNA sequences that fold into a user given secondary structure. Furthermore, constraints on the sequence can be specified, e.g. one can restrict sequence positions to a fixed nucleotide or to a set of nucleotides. Moreover, the user can allow violations of the constraints at some positions, which can be advantageous in complicated cases. The INFO-RNA web server allows biologists to design RNA sequences in an automatic manner. It is clearly and intuitively arranged and easy to use. The procedure is fast, as most applications are completed within seconds and it proceeds better and faster than other existing tools. The INFO-RNA web server is freely available at http://www.bioinf.uni-freiburg.de/Software/INFO-RNA/.

PMID:
17452349
PMCID:
PMC1933236
DOI:
10.1093/nar/gkm218
[Indexed for MEDLINE]
Free PMC Article
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18.
Nucleic Acids Res. 2006;34(17):e117. Epub 2006 Sep 20.

Using RNA secondary structures to guide sequence motif finding towards single-stranded regions.

Author information

1
Institute of Computer Science, Chair for Bioinformatics, Albert-Ludwigs-University Freiburg, Georges-Koehler-Allee 106, 79110 Freiburg, Germany.

Abstract

RNA binding proteins recognize RNA targets in a sequence specific manner. Apart from the sequence, the secondary structure context of the binding site also affects the binding affinity. Binding sites are often located in single-stranded RNA regions and it was shown that the sequestration of a binding motif in a double-strand abolishes protein binding. Thus, it is desirable to include knowledge about RNA secondary structures when searching for the binding motif of a protein. We present the approach MEMERIS for searching sequence motifs in a set of RNA sequences and simultaneously integrating information about secondary structures. To abstract from specific structural elements, we precompute position-specific values measuring the single-strandedness of all substrings of an RNA sequence. These values are used as prior knowledge about the motif starts to guide the motif search. Extensive tests with artificial and biological data demonstrate that MEMERIS is able to identify motifs in single-stranded regions even if a stronger motif located in double-strand parts exists. The discovered motif occurrences in biological datasets mostly coincide with known protein-binding sites. This algorithm can be used for finding the binding motif of single-stranded RNA-binding proteins in SELEX or other biological sequence data.

PMID:
16987907
PMCID:
PMC1903381
DOI:
10.1093/nar/gkl544
[Indexed for MEDLINE]
Free PMC Article
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19.
Bioinformatics. 2006 Aug 1;22(15):1823-31. Epub 2006 May 18.

INFO-RNA--a fast approach to inverse RNA folding.

Author information

1
Albert-Ludwigs-University Freiburg, Institute of Computer Science, Chair of Bioinformatics Georges-Koehler-Allee 106, 79110 Freiburg, Germany.

Abstract

MOTIVATION:

The structure of RNA molecules is often crucial for their function. Therefore, secondary structure prediction has gained much interest. Here, we consider the inverse RNA folding problem, which means designing RNA sequences that fold into a given structure.

RESULTS:

We introduce a new algorithm for the inverse folding problem (INFO-RNA) that consists of two parts; a dynamic programming method for good initial sequences and a following improved stochastic local search that uses an effective neighbor selection method. During the initialization, we design a sequence that among all sequences adopts the given structure with the lowest possible energy. For the selection of neighbors during the search, we use a kind of look-ahead of one selection step applying an additional energy-based criterion. Afterwards, the pre-ordered neighbors are tested using the actual optimization criterion of minimizing the structure distance between the target structure and the mfe structure of the considered neighbor. We compared our algorithm to RNAinverse and RNA-SSD for artificial and biological test sets. Using INFO-RNA, we performed better than RNAinverse and in most cases, we gained better results than RNA-SSD, the probably best inverse RNA folding tool on the market.

AVAILABILITY:

www.bioinf.uni-freiburg.de?Subpages/software.html.

PMID:
16709587
DOI:
10.1093/bioinformatics/btl194
[Indexed for MEDLINE]
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20.
Bioinformatics. 2005 Aug 1;21(15):3312-3. Epub 2005 May 26.

SECISDesign: a server to design SECIS-elements within the coding sequence.

Author information

1
Friedrich-Schiller-University Jena, Institute of Computer Science, Ernst-Abbe-Platz 2, 07743 Jena, Germany.

Abstract

SUMMARY:

SECISDesign is a server for the design of SECIS-elements and arbitrary RNA-elements within the coding sequence of an mRNA. The element has to satisfy both structure and sequence constraints. At the same time, a certain amino acid similarity to the original protein has to be kept. The designed sequence can be used for recombinant expression of selenoproteins in Escherichia coli.

AVAILABILITY:

The server is available at http://www.bio.inf.uni-jena.de/Software/SECISDesign/index.html.

PMID:
15919727
DOI:
10.1093/bioinformatics/bti507
[Indexed for MEDLINE]
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