Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants

Kim, Jong Wook; Kim, Hyung Cheol; Kim, Gyoung Mi; Yang, Jun Mo; Boeke, Jef D.; Nam, Karam

doi:10.1093/nar/28.18.3666

Cited by 38 publications

(45 citation statements)

References 19 publications

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“…For guide snoRNA processing in mammals, snoRNAs are transcribed as a part of the host gene introns. After being spliced, the excised introns are debranched by DBR1, and linearized introns are processed from both ends by exonucleases (22,34). The snoRNA region of the intron is bound by several snoRNP proteins, which protect it from degradation (10,50).…”

Section: Discussionmentioning

confidence: 99%

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Functional Association of the Microprocessor Complex with the Spliceosome

Kataoka

Fujita

Ohno

2009

Molecular and Cellular Biology

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). Based on the discovery that artificial insertion of pre-miRNAs in introns did not hamper mRNA production and that the miRNA-harboring introns were spliced more slowly than the adjacent introns, a model was previously proposed in which Drosha crops the pre-miRNA and the two cropped fragments from the pre-mRNA are subsequently trans spliced (Y. K. Kim and V. N. Kim, EMBO J. 26:775-783, 2007). However, the molecular basis for this model was not elucidated. To analyze the molecular mechanism of intronic miRNA processing, we developed an in vitro system in which both pre-miRNA processing and mRNA splicing are detected simultaneously. Our analysis using this system showed that pre-miRNA cropping from the pre-mRNA could occur kinetically faster than splicing. Glycerol gradient sedimentation experiments revealed that part of the pre-miRNA was cofractionated with the spliceosome. Furthermore, coimmunoprecipitation experiments with an anti-Drosha antibody demonstrated that Drosha was associated not only with the cropping products but also with a Y-shaped branch intron and a Y-shaped splicing intermediate. These results provide a molecular basis for the postulated existence of a pathway in which the Microprocessor complex becomes associated with the spliceosome, pre-miRNA cropping occurs prior to splicing, and trans splicing takes place between the cropped products.MicroRNAs (miRNAs) are short, noncoding RNAs that mediate posttranscriptional gene silencing via base pairing with their target mRNAs (for reviews, see references 7, 23, and 24). The majority of miRNAs are transcribed by RNA polymerase II (31) and matured through several processing steps in both the nucleus and cytoplasm (6,30,31). Primary miRNA transcripts (pri-miRNAs) are cleaved by Drosha, an RNase III-type enzyme in the nucleus (29). Drosha functions in the context of a large protein complex, termed the Microprocessor complex, that also contains DGCR8/Pasha as well as several splicing factors (8,11,13,28). Cleavage of pri-mRNAs by the Microprocessor complex results in the production of short, stem-loop-shaped RNAs called pre-miRNAs. These RNAs are subsequently exported to the cytoplasm by exportin 5 (5, 32, 52). In the cytoplasm, pre-miRNAs are cleaved by the cytoplasmic RNase III-type enzyme Dicer (4,12,16,20,26). The cleavage reaction, referred to as dicing, yields short RNA duplexes and, subsequently, incorporation of one strand of each duplex into the RNA-induced silencing complex (21, 46). The RNA-induced silencing complex binds to its target mRNAs through base pairing with the 3Ј untranslated regions and induces translational repression, mRNA cleavage, or mRNA degradation (42,49).Most miRNAs were formerly thought to have their own transcriptional units in the intergenic regions. However, recent studies revealed that about 80% of miRNAs are encoded in the intronic regions of protein-encoding or noncoding genes in mammals (25,43) and that this number is 75% in Xenopus tropicalis (48), implying that the phenomenon of the majority of miRNAs being encode...

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Section: Discussionmentioning

confidence: 99%

“…7B). RNA bands a and b were recovered from the denaturing gel and subjected to debranching with recombinant hDBR1 (22). Flag-tagged hDBR1 proteins, which were detectable by antiFlag antibody, were recovered from transfected HEK293T cells by use of anti-Flag M2 resin (Fig.…”

Section: Identification Of Rnas That Associate With Drosha During Cromentioning

confidence: 99%

Functional Association of the Microprocessor Complex with the Spliceosome

Kataoka

Fujita

Ohno

2009

Molecular and Cellular Biology

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“…Similarly, more distantly related enzymes, such as the mRNA-capping enzymes, mRNA 5Ј-triphosphatases (cluster 9) (Changela et al 2001), acid sphingomyelinase-like phosphodiesterases (cluster 8) (Testi 1996), RNA lariat debranching enzyme (Kim et al 2000), CD73-5' nucleotidase (Airas et al 1997), and MRE11A (Hopfner et al 2001) were all identified as containing protein phosphatase domains. Within the protein kinases, the guanylyl cyclases were also identified (cluster 5) (Lucas et al 2000).…”

Section: Misclassifications and Related Gene Families Detected By Trimentioning

confidence: 99%

Phosphoregulators: Protein Kinases and Protein Phosphatases of Mouse

et al. 2003

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With the completion of the human and mouse genome sequences, the task now turns to identifying their encoded transcripts and assigning gene function. In this study, we have undertaken a computational approach to identify and classify all of the protein kinases and phosphatases present in the mouse gene complement. A nonredundant set of these sequences was produced by mining Ensembl gene predictions and publicly available cDNA sequences with a panel of InterPro domains. This approach identified 561 candidate protein kinases and 162 candidate protein phosphatases. This cohort was then analyzed using TribeMCL protein sequence similarity clustering followed by CLUSTALV alignment and hierarchical tree generation. This approach allowed us to (1) distinguish between true members of the protein kinase and phosphatase families and enzymes of related biochemistry, (2) determine the structure of the families, and (3) suggest functions for previously uncharacterized members. The classifications obtained by this approach were in good agreement with previous schemes and allowed us to demonstrate domain associations with a number of clusters. Finally, we comment on the complementary nature of cDNA and genome-based gene detection and the impact of the FANTOM2 transcriptome project.

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“…The intron is then processed by exonucleolytic cleavage of the 39-lariat tail and linearization by the debranching enzyme Dbr1 ( Fig. 1D; Kim et al 2000;Cheng and Menees 2011). The spliceosome is disassembled and recycled (Arenas and Abelson 1997;Martin et al 2002).…”

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confidence: 99%

LaSSO, a strategy for genome-wide mapping of intronic lariats and branch points using RNA-seq

et al. 2014

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Both canonical and alternative splicing of RNAs are governed by intronic sequence elements and produce transient lariat structures fastened by branch points within introns. To map precisely the location of branch points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon-skipping branch points. LaSSO thus provides an effective approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated into RNA-seq pipelines. [Supplemental material is available for this article.]Introns and exons refer to noncoding and coding sequences, respectively, that constitute protein-coding genes (Gilbert 1978). To create a functional messenger RNA (mRNA), introns are excised via a highly conserved and accurate process called splicing that culminates in concatenation of exon sequences into translatable transcripts. Splicing entails two transesterification reactions catalyzed by the spliceosome, a large RNA-protein complex (Wahl et al. 2009). The first reaction involves a nucleophilic attack of an adenosine (branch point) on the 59-splice donor, resulting in a lariat structure fixed by a 29-59 phosphodiester bond; the intron remains only attached to the downstream exon (Fig. 1A,B ;Padgett et al. 1985). The second reaction involves an attack of the detached upstream exon on the 39-splice acceptor, resulting in intron lariat release and exon ligation (Fig. 1C). The intron is then processed by exonucleolytic cleavage of the 39-lariat tail and linearization by the debranching enzyme Dbr1 (Fig.

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Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants

Cited by 38 publications

References 19 publications

Functional Association of the Microprocessor Complex with the Spliceosome

Functional Association of the Microprocessor Complex with the Spliceosome

Phosphoregulators: Protein Kinases and Protein Phosphatases of Mouse

LaSSO, a strategy for genome-wide mapping of intronic lariats and branch points using RNA-seq

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