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Molecular Cloning and Functional Characterization of Factors Involved in Post-transcriptional Gene Expression
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gene expression in the eukaryotic cell is a fundamental cellular process, which consists of several distinct steps but extensively coupled to each other. From site of transcription in the nucleus to the cytoplasm, both mRNA and rRNA are associated with a proper set of proteins. These proteins influence RNA processing, transport as well as ribosome maturation. We have tried to take advantage of different model systems to understand the process of eukaryotic gene expression at the post-transcription level. To this end, we have focused on identification and characterization of several specific proteins in the context of mRNP and rRNP particles.

We have characterized a novel yeast gene MRD1, which encodes a protein with five RNA-binding domains (RBDs) and is essential for viability. Mrd1p is present in the nucleolus and the nucleoplasm. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and 40S ribosomal subunits. Mrd1p associates with the 35S prerRNA and the U3 snoRNA and is required for the initial processing of pre-rRNA at the A0-A2 sites. The presence of five RBDs in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage.

Meanwhile, an MRD1 homologue, Ct-RBD-1 with six RBDs, has also been identified and shown to involve in ribosome biogenesis in Chironomus tentans. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In the cytoplasm, Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that Ct-RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

We have characterized a novel abundant nucleolar protein, p100 in C. tentans. The p100 protein is located in the fibrillar compartment of the nucleolus, and remains in the nucleolus after digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. These results suggest that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus.

We have characterized a Dbp5 homologue in C. tentans, Ct-Dbp5. The protein becomes associated with nascent pre-mRNAs at a large number of active genes, including the Balbiani ring (BR) genes. Ct-Dbp5 is bound to nascent BR pre-mRNP particles and accompanies them through the nucleoplasm and the nuclear pore into the cytoplasm. Nuclear accumulation of Ct-Dbp5 takes place when synthesis and/or export of mRNA are inhibited. Our results indicate that most or all of the shuttling Ct-Dbp5 exiting from the nucleus associated with mRNP. Furthermore, Ct-Dbp5 is present along the mRNP fibril extending into the cytoplasm, supporting the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation.

We have shown that the export receptor CRM1 in C. tentans is associated with BR pre-mRNP while transcription takes place. We have also shown that the GTPase Ran binds to BR pre-mRNP, but its binding mainly in the interchromatin. Although both CRM1 and Ran accompany BR pre-mRNP through the nuclear pore, Leptomycin B treatment reveals that a NES-CRM1-RanGTP complex is not essential for export of the BR mRNP. Our results suggest that several export receptors associate with BR mRNP and that these receptors might have redundant functions in the nuclear export of BR mRNP.

We have analyzed four SR proteins, SC35, ASF/SF2, 9G8 and hrp45, in C. tentans. All four SR proteins genes are expressed in salivary gland cells and in several other tissues in a tissue specific pattern. We found that about 90% of all nascent pre-mRNAs bind all four SR proteins, and that approximately 10% of the pre-mRNAs associate with different subsets of the four SR proteins, suggesting that not all of four SR proteins are needed for processing of pre-mRNA. None of three examined SR proteins leave BR pre-mRNP as splicing is completed. Instead, 9G8 accompanies the mRNP to the cytoplasm, while SC35 and hrp45 leave the BR mRNP at the nuclear side of the nuclear pore complex.

Place, publisher, year, edition, pages
Inst för molekylärbiologi och funktionsgenomik , 2004. , 67 p.
Keyword [en]
gene expression, pre-mRNA processing, pre-rRNA processing
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-24ISBN: 91-7265-781-2 (print)OAI: oai:DiVA.org:su-24DiVA: diva2:191375
Public defence
2004-02-20, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00
Opponent
Supervisors
Available from: 2004-01-21 Created: 2004-01-21 Last updated: 2010-11-02Bibliographically approved
List of papers
1. Mrd1p is required for processing of pre-rRNA and for maintenance of steady-state levels of 40S ribosomal subunits in yeast
Open this publication in new window or tab >>Mrd1p is required for processing of pre-rRNA and for maintenance of steady-state levels of 40S ribosomal subunits in yeast
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2002 In: The Journal of Biological Chemistry, ISSN 1083-351X, no 277, 18431-18439 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23319 (URN)
Note
Part of urn:nbn:se:su:diva-24Available from: 2004-01-21 Created: 2004-01-21Bibliographically approved
2. A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Open this publication in new window or tab >>A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
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2002 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, 3683-3695 p.Article in journal (Refereed) Published
Abstract [en]

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

Identifiers
urn:nbn:se:su:diva-13844 (URN)10.1091/mbc.E02-03-0138 (DOI)
Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-11-02Bibliographically approved
3. A novel protein localized to the fibrillar compartment of the nucleolus and to the brush border of a secretory cell
Open this publication in new window or tab >>A novel protein localized to the fibrillar compartment of the nucleolus and to the brush border of a secretory cell
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2002 (English)In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 81, no 3, 125-137 p.Article in journal (Refereed) Published
Abstract [en]

We report the identification and molecular characterization of a novel abundant nucleolar protein of the dipteran Chironomus tentans. As shown by Western blot analysis, this protein is present in nuclear extracts in a phosphorylated form with a mobility corresponding to 100 kDa. Therefore, the protein has been termed Chironomus tentans p100, or p100 for short. Analysis of the cDNA-derived primary structure of p100 indicates a protein that contains a combination of structural domains which could be involved in interactions with proteins and nucleic acids: twelve alternating acidic and basic repeats, a glycine-arginine-rich domain and a region with two zinc fingers of the C4-type. Acidic and basic repeats are typical for a group of nonribosomal nucleolar proteins. The best-studied representatives of this group are Nopp140 and nucleolin, proteins with structural and regulatory functions in rDNA transcription. Immunocytology and immunoelectron microscopy of Chironomus tentans salivary gland cells have shown that the p100 protein is located in the fibrillar compartment of the nucleolus, while it is almost absent from the granular compartment and from the nucleoplasm. The p100 protein remains in the nucleolus after removal of RNA and DNA by digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. The presence of p100 both in the nucleolus and at the apical plasma membrane suggests that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-23321 (URN)10.1078/0171-9335-00231 (DOI)
Available from: 2004-01-21 Created: 2004-01-21 Last updated: 2012-02-16Bibliographically approved
4. The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from the gene to cytoplasm.
Open this publication in new window or tab >>The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from the gene to cytoplasm.
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2002 In: The EMBO Journal, ISSN 0261-4189, Vol. 21, no 5, 1177-1187 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23322 (URN)
Note
Part of urn:nbn:se:su:diva-24Available from: 2004-01-21 Created: 2004-01-21Bibliographically approved
5. CRM1 and Ran are present but a NES-CRM1-RanGTP complex is not required in Balbiani ring mRNP particles from the gene to the cytoplasm
Open this publication in new window or tab >>CRM1 and Ran are present but a NES-CRM1-RanGTP complex is not required in Balbiani ring mRNP particles from the gene to the cytoplasm
2004 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 117, 1553-1566 p.Article in journal (Refereed) Published
Abstract [en]

Messenger RNA is formed from precursors known as pre-mRNA. Theseprecursors associate with proteins to form pre-mRNA-protein(pre-mRNP) complexes. Processing machines cap, splice and polyadenylatethe pre-mRNP and in this way build the mRNP. These processingmachines also affect the export of the mRNP complexes from thenucleus to the cytoplasm. Export to the cytoplasm takes placethrough a structure in the nuclear membrane called the nuclearpore complex (NPC). Export involves adapter proteins in themRNP and receptor proteins that bind to the adapter proteinsand to components of the NPC. We show that the export receptorchromosomal region maintenance protein 1 (CRM1), belonging toa family of proteins known as importin-ß-like proteins,binds to gene-specific Balbiani ring (BR) pre-mRNP while transcriptiontakes place. We also show that the GTPase known as Ran bindsto BR pre-mRNP, and that it binds mainly in the interchromatin.However, we also show using leptomycin B treatment that a NES-CRM1-RanGTPcomplex is not essential for export, even though both CRM1 andRan accompany the BR mRNP through the NPC. Our results thereforesuggest that several export receptors associate with BR mRNPand that these receptors have redundant functions in the nuclearexport of BR mRNP.

Place, publisher, year, edition, pages
The Company of Biologists, 2004
Keyword
Gene expression, mRNA export, Export receptors
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-23323 (URN)10.1242/jcs.00992 (DOI)
Available from: 2004-01-21 Created: 2004-01-21 Last updated: 2012-02-16Bibliographically approved
6. Specific combinations of SR proteins are associated with gene specific nascent pre-mRNAs in vivo.
Open this publication in new window or tab >>Specific combinations of SR proteins are associated with gene specific nascent pre-mRNAs in vivo.
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23324 (URN)
Note
Part of urn:nbn:se:su:diva-24Available from: 2004-01-21 Created: 2004-01-21 Last updated: 2010-01-13Bibliographically approved

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