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Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions.

Björk P, Jin S, Zhao J, Singh OP, Persson JO, Hellman U, Wieslander L - J. Cell Biol. (2009)

Bottom Line: However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences.All four SR proteins are associated with the BR mRNPs during export to the cytoplasm.ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91 Stockholm, Sweden.

ABSTRACT
Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3' end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo.

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Different types of SR proteins are associated with BR pre-mRNPs. (A–D) Chromosome IV was stained with anti-SC35 (A), anti-ASF/SF2 (B), anti-hrp45/SRp55 (C), or anti-9G8 (D) antibodies and analyzed by EM. Gold particles attached to the secondary antibodies show specific labeling of nascent BR pre-mRNPs (arrows). (E and F) Combinations of anti-SC35 (6-nm gold) and anti-9G8 (12-nm gold) antibodies (E) and anti-hrp45/SRp55 (6-nm gold) and anti-9G8 (12-nm gold) antibodies (F) showed that individual BR pre-mRNPs were associated with these combinations of SR proteins. Two separate examples are shown for each combination of antibodies. A schematic representation of the double-labeled individual BR pre-mRNP is shown to the right of each micrograph. Bars: (A–D) 200 nm; (E and F) 100 nm.
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fig3: Different types of SR proteins are associated with BR pre-mRNPs. (A–D) Chromosome IV was stained with anti-SC35 (A), anti-ASF/SF2 (B), anti-hrp45/SRp55 (C), or anti-9G8 (D) antibodies and analyzed by EM. Gold particles attached to the secondary antibodies show specific labeling of nascent BR pre-mRNPs (arrows). (E and F) Combinations of anti-SC35 (6-nm gold) and anti-9G8 (12-nm gold) antibodies (E) and anti-hrp45/SRp55 (6-nm gold) and anti-9G8 (12-nm gold) antibodies (F) showed that individual BR pre-mRNPs were associated with these combinations of SR proteins. Two separate examples are shown for each combination of antibodies. A schematic representation of the double-labeled individual BR pre-mRNP is shown to the right of each micrograph. Bars: (A–D) 200 nm; (E and F) 100 nm.

Mentions: The immunofluorescence results, including sensitivity to RNase, suggested that each individual transcript binds multiple SR proteins. The association of the SR proteins with BR1 and BR2 pre-mRNPs was further investigated by immuno-EM. The BR1 and BR2 pre-mRNAs associate with various RNA-binding proteins cotranscriptionally and gradually form morphologically characteristic pre-mRNA–protein complexes (Daneholt, 2001), which are referred to as BR pre-mRNPs (see Fig. 4 B). We found that the SR proteins were associated with BR pre-mRNPs at all stages of transcription and packaging. Growing BR pre-mRNPs labeled for each SR protein are shown in Fig. 3 (A–D).


Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions.

Björk P, Jin S, Zhao J, Singh OP, Persson JO, Hellman U, Wieslander L - J. Cell Biol. (2009)

Different types of SR proteins are associated with BR pre-mRNPs. (A–D) Chromosome IV was stained with anti-SC35 (A), anti-ASF/SF2 (B), anti-hrp45/SRp55 (C), or anti-9G8 (D) antibodies and analyzed by EM. Gold particles attached to the secondary antibodies show specific labeling of nascent BR pre-mRNPs (arrows). (E and F) Combinations of anti-SC35 (6-nm gold) and anti-9G8 (12-nm gold) antibodies (E) and anti-hrp45/SRp55 (6-nm gold) and anti-9G8 (12-nm gold) antibodies (F) showed that individual BR pre-mRNPs were associated with these combinations of SR proteins. Two separate examples are shown for each combination of antibodies. A schematic representation of the double-labeled individual BR pre-mRNP is shown to the right of each micrograph. Bars: (A–D) 200 nm; (E and F) 100 nm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654125&req=5

fig3: Different types of SR proteins are associated with BR pre-mRNPs. (A–D) Chromosome IV was stained with anti-SC35 (A), anti-ASF/SF2 (B), anti-hrp45/SRp55 (C), or anti-9G8 (D) antibodies and analyzed by EM. Gold particles attached to the secondary antibodies show specific labeling of nascent BR pre-mRNPs (arrows). (E and F) Combinations of anti-SC35 (6-nm gold) and anti-9G8 (12-nm gold) antibodies (E) and anti-hrp45/SRp55 (6-nm gold) and anti-9G8 (12-nm gold) antibodies (F) showed that individual BR pre-mRNPs were associated with these combinations of SR proteins. Two separate examples are shown for each combination of antibodies. A schematic representation of the double-labeled individual BR pre-mRNP is shown to the right of each micrograph. Bars: (A–D) 200 nm; (E and F) 100 nm.
Mentions: The immunofluorescence results, including sensitivity to RNase, suggested that each individual transcript binds multiple SR proteins. The association of the SR proteins with BR1 and BR2 pre-mRNPs was further investigated by immuno-EM. The BR1 and BR2 pre-mRNAs associate with various RNA-binding proteins cotranscriptionally and gradually form morphologically characteristic pre-mRNA–protein complexes (Daneholt, 2001), which are referred to as BR pre-mRNPs (see Fig. 4 B). We found that the SR proteins were associated with BR pre-mRNPs at all stages of transcription and packaging. Growing BR pre-mRNPs labeled for each SR protein are shown in Fig. 3 (A–D).

Bottom Line: However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences.All four SR proteins are associated with the BR mRNPs during export to the cytoplasm.ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91 Stockholm, Sweden.

ABSTRACT
Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3' end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo.

Show MeSH