Limits...
An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast.

Long RM, Gu W, Meng X, Gonsalvez G, Singer RH, Chartrand P - J. Cell Biol. (2001)

Bottom Line: LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA.Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm.We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

View Article: PubMed Central - PubMed

Affiliation: Medical College of Wisconsin, Department of Microbiology and Molecular Genetics, Milwaukee, Wisconsin 53226, USA. rlong@mcw.edu

ABSTRACT
The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

Show MeSH

Related in: MedlinePlus

A band mobility shift assay identifies proteins that specifically bind the E3 element. Radiolabeled E3 transcripts were incubated with yeast protein extracts, and then with RNase T1 and heparin at room temperature. RNA–protein complexes were resolved in 4% native gel and visualized by autoradiography. Lane 1, E3 transcripts incubated with K699 extract; lanes 2 and 3, E3 transcripts incubated with K699 extracts in the presence of 200× molar excess of pGEM RNA and unlabeled E3 transcripts, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169461&req=5

Figure 1: A band mobility shift assay identifies proteins that specifically bind the E3 element. Radiolabeled E3 transcripts were incubated with yeast protein extracts, and then with RNase T1 and heparin at room temperature. RNA–protein complexes were resolved in 4% native gel and visualized by autoradiography. Lane 1, E3 transcripts incubated with K699 extract; lanes 2 and 3, E3 transcripts incubated with K699 extracts in the presence of 200× molar excess of pGEM RNA and unlabeled E3 transcripts, respectively.

Mentions: To verify that RNA-binding proteins required for ASH1 mRNA localization existed, we used the localization element E3 in a gel mobility assay with yeast extracts (Fig. 1). The gel shift revealed an RNA-specific band which could not be competed with heterologous RNA. We next used the element as an affinity ligand to isolate binding proteins; however, the proteins isolated by this approach were not specific, being mainly ribosomal proteins (data not shown). Consequently, we initiated an in vivo approach for the identification of RNA-binding proteins required for ASH1 mRNA localization. We used the three-hybrid system, which is a molecular genetic approach for the identification and characterization of RNA-binding proteins in vivo (SenGupta et al. 1996). In this assay, formation of a specific RNA–protein complex in yeast results in transcriptional activation of the reporter genes HIS3 and lacZ.


An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast.

Long RM, Gu W, Meng X, Gonsalvez G, Singer RH, Chartrand P - J. Cell Biol. (2001)

A band mobility shift assay identifies proteins that specifically bind the E3 element. Radiolabeled E3 transcripts were incubated with yeast protein extracts, and then with RNase T1 and heparin at room temperature. RNA–protein complexes were resolved in 4% native gel and visualized by autoradiography. Lane 1, E3 transcripts incubated with K699 extract; lanes 2 and 3, E3 transcripts incubated with K699 extracts in the presence of 200× molar excess of pGEM RNA and unlabeled E3 transcripts, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169461&req=5

Figure 1: A band mobility shift assay identifies proteins that specifically bind the E3 element. Radiolabeled E3 transcripts were incubated with yeast protein extracts, and then with RNase T1 and heparin at room temperature. RNA–protein complexes were resolved in 4% native gel and visualized by autoradiography. Lane 1, E3 transcripts incubated with K699 extract; lanes 2 and 3, E3 transcripts incubated with K699 extracts in the presence of 200× molar excess of pGEM RNA and unlabeled E3 transcripts, respectively.
Mentions: To verify that RNA-binding proteins required for ASH1 mRNA localization existed, we used the localization element E3 in a gel mobility assay with yeast extracts (Fig. 1). The gel shift revealed an RNA-specific band which could not be competed with heterologous RNA. We next used the element as an affinity ligand to isolate binding proteins; however, the proteins isolated by this approach were not specific, being mainly ribosomal proteins (data not shown). Consequently, we initiated an in vivo approach for the identification of RNA-binding proteins required for ASH1 mRNA localization. We used the three-hybrid system, which is a molecular genetic approach for the identification and characterization of RNA-binding proteins in vivo (SenGupta et al. 1996). In this assay, formation of a specific RNA–protein complex in yeast results in transcriptional activation of the reporter genes HIS3 and lacZ.

Bottom Line: LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA.Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm.We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

View Article: PubMed Central - PubMed

Affiliation: Medical College of Wisconsin, Department of Microbiology and Molecular Genetics, Milwaukee, Wisconsin 53226, USA. rlong@mcw.edu

ABSTRACT
The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

Show MeSH
Related in: MedlinePlus