Molecular structure and intracellular location of five RNA : associated proteins in the salivary glands of the dipteran Chironomus tentans

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Cell and Molecular Biology

Sammanfattning: The structure and intracellular flow of specific RNA-associated proteins have been characterized in the salivary gland cells in the dipteran Chironomus tentans. These cells contain four polytene chromosomes (I-IV), two of which harbour a nucleolus. The chromosomal sites active in pre-messenger RNA transcription are called chromosomal puffs; some of the puffs are especially conspicuous and are designated Balbiani rings (BRs). The pre-messenger ribonucleoprotein (RNP) particles in BRs 1 and 2 can be visualized in the electron microscope during assembly on the BR genes and during transport to and through the nuclear pores. In the present studies, monoclonal antibodies were raised against single-stranded DNA binding proteins extracted from nuclei of C. tentans tissue culture cells. The antibodies were used to select cDNA clones from C. tentans expression libraries, and subsequent DNA sequence analysis revealed the primary structure of the proteins. The antibodies were also applied in immunocytology and immunoelectron microscopy studies. Three proteins, hrp36, hrp45 and hrp23, are present in BR 1-2 pre-mRNP particles. The hrp36 protein was shown to contain two RNA-binding domains and an auxiliary glycine-rich domain. The protein is added to the BR transcript concomitant with transcription and remains associated with the transcript in the nucleoplasm, during the transport through the nuclear pores, and even during protein synthesis in the cytoplasm. These observations strongly suggested that hrp36 is involved in RNA export. The hip45 protein has two RNA-binding domains and an auxiliary domain rich in serine and arginine. The protein proved to be homologous to some members of the SR protein family. The hrp45 protein reaches the nuclear pore complex bound to the RNA, but when the transcript enters the central channel of the nuclear pore complex, the protein is released from the particle. The results suggested that hrp45 acts as a structural protein bound to exonic RNA during splicing and could exert additional functions. The hrp23 protein has only one RNA-binding domain and its auxiliary domain is rich in glycine, arginine and serine. The hrp23 protein is bound to the growing BR transcripts and is present in the BR RNP particles in the nucleoplasm. However, hrp23 could not be detected in the BR particles bound to the nuclear pore complex, indicating that the hrp23 is released just prior to or at the binding of the particles to the pore complex. Taken together, the studies of the pre-mRNA binding proteins hrp36, hrp45 and hrp23 show that each protein exhibits a distinct flow pattern, probably related to the particular function of each protein in the maturation andlor transport of BR pre-mRNA. A BR3-specific protein, hrp130, was shown to be a new protein with no known RNA-binding sequence motif. Its specific chromosomal location indicated that hrpl30 binds to RNA with high sequence preference. The nucleolar 95 kD protein proved to be a phosphoprotein located in the fibrillar portion of the nucleoli. The amino-terminal and central part of the protein consists of alternating acidic and basic regions, and the carboxy-terminal part contains frequent arginine- glycine-glycine or phenylalanine-glycine-glycine tripeptides. The 95 kD protein shows striking similarities to the vertebrate nucleolar proteins nucleolin and Nopp140.

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