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Non-protein-coding RNA: Transcription and regulation of ribosomal RNA
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Ann-Kristin Östlund Farrants)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized.

In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network.

Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2014. , 58 p.
Keyword [en]
ribosomal RNA, non-coding RNA, ribosomal genes, rDNA gene, B-WICH, chromatin remodelling, histone modification
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-102718ISBN: 978-91-7447-906-5 (print)OAI: oai:DiVA.org:su-102718DiVA: diva2:712890
Public defence
2014-05-23, Lecture hall E306, Arrheniuslaboratorierna, Svante Arrhenius Väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript

Available from: 2014-04-29 Created: 2014-04-16 Last updated: 2014-04-29Bibliographically approved
List of papers
1. The Chromatin Remodelling Complex B-WICH Changes the Chromatin Structure and Recruits Histone Acetyl-Transferases to Active rRNA Genes
Open this publication in new window or tab >>The Chromatin Remodelling Complex B-WICH Changes the Chromatin Structure and Recruits Histone Acetyl-Transferases to Active rRNA Genes
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 4, e19184Article in journal (Refereed) Published
Abstract [en]

The chromatin remodelling complex B-WICH, which comprises the William syndrome transcription factor (WSTF), SNF2h, and nuclear myosin 1 (NM1), is involved in regulating rDNA transcription, and SiRNA silencing of WSTF leads to a reduced level of 45S pre-rRNA. The mechanism behind the action of B-WICH is unclear. Here, we show that the B-WICH complex affects the chromatin structure and that silencing of the WSTF protein results in a compaction of the chromatin structure over a 200 basepair region at the rRNA promoter. WSTF knock down does not show an effect on the binding of the rRNA-specific enhancer and chromatin protein UBF, which contributes to the chromatin structure at active genes. Instead, WSTF knock down results in a reduced level of acetylated H3-Ac, in particular H3K9-Ac, at the promoter and along the gene. The association of the histone acetyl-transferases PCAF, p300 and GCN5 with the promoter is reduced in WSTF knock down cells, whereas the association of the histone acetyl-transferase MOF is retained. A low level of H3-Ac was also found in growing cells, but here histone acetyl-transferases were present at the rDNA promoter. We propose that the B-WICH complex remodels the chromatin structure at actively transcribed rRNA genes, and this allows for the association of specific histone acetyl-transferases.

National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-68540 (URN)10.1371/journal.pone.0019184 (DOI)000290024700094 ()
Note

7

Available from: 2012-01-04 Created: 2012-01-04 Last updated: 2017-12-08Bibliographically approved
2. The B-WICH chromatin-remodelling complex initiates the regulation of RNA polymerase III by c-Myc
Open this publication in new window or tab >>The B-WICH chromatin-remodelling complex initiates the regulation of RNA polymerase III by c-Myc
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Transcription by RNA polymerase III in eukaryotic cells is closely associated with cell growth and proliferation, and regulated by several proliferative signals. In addition, the chromatin-remodelling complex B-WICH, comprised of William syndrome transcription factor, the ATPase SNF2h and nuclear myosin, binds to the 5S rRNA and 7SL genes and activates transcription, but the mechanism behind is poorly understood. Here, we have used high‑resolution MN walking to show that the role of B-WICH in RNA polymerase III transcription is to induce local alterations of the chromatin structure in the vicinity of the 5S rRNA and 7SL RNA genes. In the 5S rDNA, the remodelled region harbours an E-box, to which c-Myc, together with Max, binds in a B-WICH dependent way.  Both B-WICH and c-Myc are required for the subsequent histone acetylation of histone H3. Our results present two ways for c-Myc to alter 5S rRNA transcription; to bind to the RNA polymerase III machinery at the promoter and to an E-box in the intergenic spacer. We propose a model in which the B-WICH complex is required to maintain an open chromatin structure at these RNA polymerase III genes, which is a prerequisite for other regulatory factors to bind at the gene.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-103000 (URN)
Available from: 2014-04-25 Created: 2014-04-25 Last updated: 2016-01-29Bibliographically approved
3. Non-coding RNAs from the rDNA intergenic repeat are transcribed by RNA polymerase I and II and have different functions
Open this publication in new window or tab >>Non-coding RNAs from the rDNA intergenic repeat are transcribed by RNA polymerase I and II and have different functions
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Long intergenic non-coding RNA, linc RNA, are often produced from intergenic sequences and have been ascribed diverse functions, such as regulating mRNA levels and being involved in the formation of heterochromatin. We show here that the intergenic spacer region (IGS) of the ribosomal DNA gene repeat in human cells is transcribed. Three ncRNAs, the IGS19asRNA, the IGS32asRNA and the IGS38RNA, of 500, 800 and 1300 bases, respectively, were isolated and investigated. Two of them, the IGS19asRNA and the IGS32asRNA, were transcribed in the antisense direction with respect to the rRNA and in the sense direction for the IGS38RNA. We also showed that the ncRNAs were transcribed by different RNA polymerases; the IGS19asRNA and the IGS38RNA were transcribed by RNA polymerase II and the IGS32asRNA were transcribed by RNA polymerase I. The three ncRNAs were also differentially regulated; IGS19asRNA induced upon heat shock and the level of the IGS32asRNA increased upon glucose feeding, similar to the 45S rRNA. In addition, the ncRNAs IGS19asRNA and IGS32asRNA were found at different locations in the nucleus, with IGS19asRNA located in a speckled pattern in the nucleus and IGS32asRNA associated with chromatin bound to heterochromatin protein 1. This suggests that the IGS32asRNA has a role in heterochromatin formation.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-103001 (URN)
Available from: 2014-04-25 Created: 2014-04-25 Last updated: 2016-01-29Bibliographically approved
4. Nuclear Myosin 1c Facilitates the Chromatin Modifications Required to Activate rRNA Gene Transcription and Cell Cycle Progression
Open this publication in new window or tab >>Nuclear Myosin 1c Facilitates the Chromatin Modifications Required to Activate rRNA Gene Transcription and Cell Cycle Progression
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2013 (English)In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 9, no 3, e1003397Article in journal (Refereed) Published
Abstract [en]

Actin and nuclear myosin 1c (NM1) cooperate in RNA polymerase I (pol I) transcription. NM1 is also part of a multiprotein assembly, B-WICH, which is involved in transcription. This assembly contains the chromatin remodeling complex WICH with its subunits WSTF and SNF2h. We report here that NM1 binds SNF2h with enhanced affinity upon impairment of the actin-binding function. ChIP analysis revealed that NM1, SNF2h, and actin gene occupancies are cell cycle-dependent and require intact motor function. At the onset of cell division, when transcription is temporarily blocked, B-WICH is disassembled due to WSTF phosphorylation, to be reassembled on the active gene at exit from mitosis. NM1 gene knockdown and motor function inhibition, or stable expression of NM1 mutants that do not interact with actin or chromatin, overall repressed rRNA synthesis by stalling pol I at the gene promoter, led to chromatin alterations by changing the state of H3K9 acetylation at gene promoter, and delayed cell cycle progression. These results suggest a unique structural role for NM1 in which the interaction with SNF2h stabilizes B-WICH at the gene promoter and facilitates recruitment of the HAT PCAF. This leads to a permissive chromatin structure required for transcription activation.

National Category
Genetics
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-89730 (URN)10.1371/journal.pgen.1003397 (DOI)000316866700068 ()
Funder
Swedish Research CouncilSwedish Cancer Society
Note

AuthorCount:10;

Available from: 2013-05-07 Created: 2013-05-06 Last updated: 2017-12-06Bibliographically approved

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