Founded in 1975 as the all Union Research Institute of Applied Enzimology, currently,  the Institute of Biotechnology is mainly involved in research and training in the fields of biotechnology and molecular biology, including research and development of recombinant biomedical proteins, genetic and molecular studies of restriction modification phenomenon, developing of viruses diagnostics, epigenetic study of small RNA, drug design and synthesis, bioinformatics.

Regulation of gene expression by alternative splicing

Spausdinti

Regulation of hypoxia-inducible factor HIF-3α expression via the alternative pre-mRNA splicing mechanism (Dr. A. Kanopka)


Recent genome-wide analyses of alternative splicing indicate that up to 70% of human genes may have alternative splice forms, suggesting that alternative splicing together with various posttranslational modifications plays a major role in the production of proteome complexity. Splice-site selection under normal physiological conditions is regulated in the developmental stage in a tissue type-specific manner by changing the concentrations and the activity of splicing regulatory proteins.
Changes in splice-site selection have been observed in various types of cancer and may affect genes implicated in tumor progression and in susceptibility to cancer. Splicing defects can arise from inherited or somatic mutations in cis-acting regulatory elements (splice donor, acceptor and branch sites, and exonic and intronic splicing enhancers and silencers) or variations in the composition, concentration, localization, and activity of regulatory proteins. This may lead to altered efficiency of splice-site recognition, resulting in overexpression or down-regulation of certain splice variants, a switch in splice-site usage, or failure to recognize splice sites correctly, resulting in cancer-specific splice forms. At least in some cases, changes in splicing have been shown to play a functionally significant role in tumorigenesis, either by inactivating tumor suppressors or by gain of function of proteins promoting tumor development. Thus, the identification of cancer specific splice forms provides a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention.
Hypoxia has long been recognized as a common feature of solid tumors and a negative prognostic factor for response to treatment and survival of cancer patients. The discovery of hypoxia-inducible factor 1 (HIF-1), a molecular determinant of the response of mammalian cells to hypoxia, has led to the identification of a ‘‘molecular target’’ of hypoxia suitable for the development of cancer therapeutics. Immunohistochemical analyses using monoclonal antibodies revealed that HIF-a is overexpressed in the majority of human cancers, where it is associated with patient mortality and poor response to treatment. As it is established HIF-1 role in cancer biology is complex. HIF-1 inhibitors should be incorporated in combination strategies to effectively target multiple cellular components of the tumor microenvironment and redundant signaling pathways frequently deregulated in human cancer.
The role of other two HIFa subunits (HIF-2a and HIF-3a), in different tumor types, still remains to be defined.
A splice variant of HIF-3, inhibitory PAS domain protein (IPAS), inhibits the dimerization of HIF-1 and ARNT. This protein is translated by an alternatively spliced variant of mouse HIF-3 mRNA, which shares exons 2 – 6 with HIF-3, but which contains exons 1 and 7 different from those of the wild-type mRNA. Since all exons including exons 1 and 7 are present near the Hif 3 locus, this variant must be a spliced mRNA transcribed by the Hif 3 gene. IPAS protein contains a bHLH domain and a PAS domain, which are the common structures present in the HIF family. However, it has no transactivation function due to its lack of a transactivation domain, but does dominantly and negatively regulate HIF-mediated gene expression. IPAS expression in hepatoma cells selectively impairs the induction of hypoxia-inducible genes regulated by HIF-1 and results in retarded tumor growth and tumor vascular density in vivo. In mice, IPAS was selectively expressed in Purkinje cells of the cerebellum and in the corneal epithelium of the eye. Moreover, the expression of IPAS in the cornea correlates with low VEGF gene expression under hypoxic conditions.
We established that essential splicing factors SR proteins are involved in dependable from oxygen tension pre-mRNA splicing regulation.
The identification of selective HIF-1 inhibitors which will reprogram HIF-1 function would not only be useful for the potential therapeutic implications but also for their application as analytic tools.
This work was supported by the EU Framework 6 Programme (project Euroxy).

Partners:
Prof. L.Poellinger, Karolinska Institute, Stockholm, Sweden
Dr. J.Makino, Tokyo University, Tokyo, Japan
Prof. E. Pettersen, Oslo University, Oslo, Norway
Prof. P.Ebbesen, Aalborg University, Aalborg, Denmark
Prof.A.C. Cato, Karlsruhe University, Karlsruhe, Germany

Grants:
EU Framework 6 Programme
Lithuanian State Science and Studies Foundation
NorFa network