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Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation
S R Valentini1, V H Weiss, P A Silver
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. valentsr@fcfar.unesp.br
Abstract:
Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrplp is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrpl p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrpl p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.
Insights
Methylation does not affect the binding of heterogeneous ribonucleoprotein (Hrp1p) to specific RNA sequences. However, RNA binding inhibits Hrp1p methylation, suggesting methylation precedes RNA binding in yeast cells.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- Hrp1p is a yeast heterogeneous ribonucleoprotein (hnRNP) crucial for mRNA 3'-end processing and export.
- Arginine methylation is a post-translational modification affecting RNA-binding proteins, including Hrp1p.
- Specific RNA sequences, like UAUAUA, are high-affinity binding sites for Hrp1p.
Purpose of the Study:
- To investigate the impact of arginine methylation on Hrp1p's specific RNA binding.
- To determine the effect of RNA binding on Hrp1p methylation by Hmt1p.
- To elucidate the temporal order of Hrp1p methylation and RNA binding.
Main Methods:
- Purification of functional recombinant Hrp1p and Hmt1p.
- Identification of high-affinity RNA binding sites for Hrp1p using functional recombinant protein.
- In vitro methylation of Hrp1p by Hmt1p.
- Assessment of RNA binding affinity for both methylated and unmethylated Hrp1p.
- Analysis of the effect of RNA on Hrp1p methylation.
Main Results:
- Hrp1p specifically binds to UAUAUA sequences, known efficiency elements for mRNA 3'-end formation.
- Methylated Hrp1p exhibits unchanged high-affinity binding to UAUAUA-containing RNAs compared to unmethylated Hrp1p.
- RNA binding inhibits Hrp1p methylation, with this inhibition being stronger for RNAs that Hrp1p binds specifically.
- These findings indicate that methylation does not alter Hrp1p's specific RNA recognition.
Conclusions:
- Protein methylation of Hrp1p does not influence its specific RNA binding affinity.
- RNA binding to Hrp1p inhibits its subsequent methylation.
- A model is proposed where Hrp1p methylation occurs in the nucleus before its association with RNA.