[Protein complementation as tool for studying protein-protein interactions in living cells].

Association and degradation of protein complexes play essential role in a majority of normal and pathologic processes, which take place in living cell. Studying the underlying mechanisms of those interactions would give deeper understanding of specific causes of disease progression and would allow developing new therapeutic strategies. The majority of technical approaches currently used for detecting protein association include in vitro protein extraction and purification, whereas more relevant results require methods that can be used in vivo. One of a few approaches for in vivo protein association detection is based on reporter protein fragment complementation. Reporter systems based on protein complementation rely on reconstitution of reporter protein fluorescent or enzymatic activity which occurs upon reassociation of protein fragments and could be measured by colorimetry, luminometry or fluorimetry. Protein complementation is widely used to develop reporter systems for analysis of protein interactions, for functional dissection of signal transduction pathways and for performing high-throughput screenings to discover new protein interaction partners. Currently developed approaches that utilize protein fragment complementation have possibilities that extend far beyond simple detection of interaction in a pair of proteins.

[A lentivirus vector based assay system for quantitative detection of intracellular translocations of recombinant proteins].

An enzymatic assay system is described that allows quantitative localization within different cellular structures of recombinant proteins. The system is based on alpha-complementation of beta-galactosidase. The large omega-fragment of beta-galactosidase is expressed in predefined cellular structures with the aid of attached protein localization signals. The obtained reporter cell lines are used for the introduction of a second construct that expresses a protein of study fused with a shorter alpha-fragment of beta-galactosidase. Physical proximity of the two recombinant proteins carrying beta-galactosidase fragments results in reconstitution of an active enzyme, and the activity can be measured in a plate reader. The recombinant constructs are based on lentiviral vectors, which allows rapid and efficient introduction of recombinant proteins into cells by infection with stocks of lentiviral particles. The efficiency of the system is demonstrated with transcriptional factor FOXO3A, which is shuttling between cytoplasm and nuclei in model colon carcinoma cell line RKO.

[Alternative transcripts from POLRMT responsible for synthesis of nuclear RNA polymerase IV].

By analyzing ESTs that correspond to human POLRMT gene encoding mitochondrial RNA polymerase (mtRNAP) we revealed an alternatively spliced transcript. We confirmed the existence of the transcript that contain additional 225 nucleotides from proximal part of intron 1 by RT-PCR using RNA from HeLa cells. In mouse and rat there are similar alternative transcripts that contain entire intron 1 sequences. In addition, in mouse we revealed third transcript that contain extra exon derived from 142 bp of intron 2. The revealed alternative transcripts, in contrast to the mRNA encoding mtRNAP, specify N-terminally truncated protein lacking mitochondrial targeting signal. This protein has strictly nuclear localization and corresponds to nuclear RNA polymerase IV that we recently identified.