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Characterization of RNA polymerase II subunit Rpb7 in silencing and transcription
Södertörn University, School of Life Sciences. Karolinska Institutet.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The DNA in eukaryotes is arranged in fibres of chromatin. The chromatin may be more or less compacted and the degree of condensation of the chromatin affects the accessibility of the DNA. The accessibility of the DNA, in turn, affects transcription and gene regulation. Genes within inaccessible DNA are commonly repressed whereasgenes within accessible DNA are active and expressed. This thesis concerns the interplay between chromatin and transcription with focus on the function of the RNA polymerase II (pol II) subunit Rpb7. We have demonstrated that processing of centromeric transcripts by the ribonuclease III family protein Dcr1 is required for heterochromatin formation at the centromeres of Schizosaccharomyces pombe. A point mutation in the pol II subunit Rpb7 caused a specific defect in centromeric heterochromatin formation. We have shown i) that the centromeric transcripts that accumulate in dcr1delta cells are products of pol II, ii) the rpbG150D mutation is deficient in recognition and/or initiation of transcription from the centromeric promoter. Transcription by pol II within the centromeres was surprising since insertion of marker genes within these loci normally results in repression of pol II transcription. Here, paradoxically, pol II transcription was required for the construction of the inaccessible heterochromatin structure. Our analysis of sRNA in S. pombe revealed that most centromeric siRNA are originating from two clusters, which are repeated several times within the centromeres. This lead us to propose a model in which centromeric transcripts fold into double stranded structures that are processed by Dcr1. The resulting siRNAs may contribute with the starting signal for the RNAi feedback loop required for heterochromatin formation at the centromeres. Finally, we demonstrate that the genome-wide association of Rpb7 is nearly identical to that of the core pol II subunit Rpb2, indicating a general role for Rpb7 in transcription. We further show that the occupancy pattern of Rpb4, a pol II subunit that forms a subcomplex together with Rpb7, differs from those of Rpb2 and Rpb7. Rpb4 may therefore have a less general function in transcription than Rpb7. Hence, transcription by pol II is required not only for gene expression but also for repression via formation of inaccessible heterochromatin.

Place, publisher, year, edition, pages
Stockholm: Karolinska Institutet , 2009. , 55 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:sh:diva-31550ISBN: 978-91-7409-606-4 (print)OAI: oai:DiVA.org:sh-31550DiVA: diva2:1060743
Public defence
2009-12-04, Föreläsningssalen, plan 4, NOVUM, Huddinge, 10:00
Opponent
Supervisors
Available from: 2016-12-29 Created: 2016-12-29 Last updated: 2016-12-29Bibliographically approved
List of papers
1. Dicer is required for chromosome segregation and gene silencing in fission yeast cells
Open this publication in new window or tab >>Dicer is required for chromosome segregation and gene silencing in fission yeast cells
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2002 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 99, no 26, 16648-16653 p.Article in journal (Refereed) Published
Abstract [en]

RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1(+)) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into approximate to23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1(+) was required for silencing of two genes containing a conserved motif.

National Category
Natural Sciences
Identifiers
urn:nbn:se:sh:diva-15719 (URN)10.1073/pnas.212633199 (DOI)000180101600028 ()12482946 (PubMedID)2-s2.0-0037168521 (ScopusID)
Available from: 2012-03-08 Created: 2012-03-06 Last updated: 2016-12-29Bibliographically approved
2. RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing
Open this publication in new window or tab >>RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing
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2005 (English)In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 19, no 19, 2301-2306 p.Article in journal (Refereed) Published
Abstract [en]

Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in presiRNA transcription. We define a centromeric presiRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-14442 (URN)10.1101/gad.344205 (DOI)000232433000008 ()1240039 (PubMedID)2-s2.0-25844480848 (ScopusID)
Available from: 2012-01-31 Created: 2011-12-23 Last updated: 2016-12-29Bibliographically approved
3. Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA
Open this publication in new window or tab >>Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA
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2009 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 28, no 24, 3832-3844 p.Article in journal (Refereed) Published
Abstract [en]

The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 50-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. The EMBO Journal (2009) 28, 3832-3844. doi: 10.1038/emboj.2009.351; Published online 26 November 2009

Keyword
centromeres, RNAi, small RNA, S. pombe
National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:sh:diva-17674 (URN)10.1038/emboj.2009.351 (DOI)000272833700006 ()19942857 (PubMedID)2-s2.0-72449159549 (ScopusID)
Funder
Swedish Cancer SocietySwedish Research CouncilEU, European Research Council, LSHGCT-2004-503433
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2012-12-14 Created: 2012-12-14 Last updated: 2016-12-29Bibliographically approved
4. Differential Genome-wide Occupancies of RNA Polymerase II Subunits Rpb4 and Rpb7 in Fission Yeast
Open this publication in new window or tab >>Differential Genome-wide Occupancies of RNA Polymerase II Subunits Rpb4 and Rpb7 in Fission Yeast
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31549 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2016-12-29 Created: 2016-12-29 Last updated: 2016-12-29Bibliographically approved

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