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HEL/UAP56 Binds Cotranscriptionally to the Balbiani Ring Pre-mRNA in an Intron-Independent Manner and Accompanies the BR mRNP to the Nuclear Pore
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2002 (English)In: Current Biology, ISSN 0960-9822, Vol. 12, no 10, 859-62 p.Article in journal (Refereed) Published
Abstract [en]

The splicing factor UAP56/HEL/Sub2p is essential for mRNA export [1], [2], [3] and [4]. It has been proposed [1] and [2] that UAP56/HEL/Sub2p interacts with the pre-mRNA during splicing and recruits the export factor Aly/REF/Yra1 (reviewed in [5]) to the spliced mRNA. However, UAP56/HEL/Sub2p also participates in the transport of intronless mRNAs, and thus its role in export is not necessarily coupled to splicing [2], [3] and [4]. Here, we characterize the HEL protein of Chironomus tentans and we analyze in situ the interaction of HEL with a natural export substrate, the Balbiani ring pre-messenger ribonucleoprotein (BR pre-mRNP, reviewed in [6]). Using immunoelectron microscopy, we show that HEL binds to the BR pre-mRNP cotranscriptionally and that incorporation of HEL into the pre-mRNP is independent of the location of introns along the BR pre-mRNA. We also show that HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation to the cytoplasm. Aly/REF is also released from the BR mRNP during translocation but after dissociation of HEL. In summary, we have shown that binding of HEL to the BR pre-mRNA occurs independently of splicing, and we have established the point in the export pathway at which HEL and Aly/REF interact with the mRNP.

Place, publisher, year, edition, pages
Current Biology Limited , 2002. Vol. 12, no 10, 859-62 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-23275DOI: 10.1016/S0960-9822(02)00840-0OAI: oai:DiVA.org:su-23275DiVA: diva2:191083
Note
Part of urn:nbn:se:su:diva-218Available from: 2004-08-30 Created: 2004-08-30 Last updated: 2010-01-08Bibliographically approved
In thesis
1. Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans
Open this publication in new window or tab >>Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of Chironomus tentans provide a suitable model system for studying gene expression in situ, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in C. tentans named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of C. tentans salivary gland cells.

Hrp59 is a C. tentans homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in Drosophila cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences.

Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1.

Hrp65 binds to HEL/UAP56 in C. tentans cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik, 2004. 80 p.
Keyword
gene expression, RNA-binding proteins, mRNA biogenesis, nuclear structure
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-218 (URN)91-7265-921-1 (ISBN)
Public defence
2004-09-20, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 09:15
Opponent
Supervisors
Available from: 2004-08-30 Created: 2004-08-30Bibliographically approved

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