Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3'-end formation

Area of Science:

• Molecular Biology
• RNA Processing
• Gene Expression Regulation

Background:

• Maturation of the 3' end of mRNA in Saccharomyces cerevisiae requires four factors: cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP).
• CF I and CF II are involved in mRNA cleavage, while PAP and PF I, along with CF II subunits, participate in polyadenylation.
• Two sequences, an A-rich and a UA-rich element, direct these factors to the correct site on the mRNA.

Purpose of the Study:

• To elucidate the specific RNA binding interactions of Rna15 within the CF I complex during mRNA 3' end processing.
• To determine the role of Rna15 and its interaction with other proteins and RNA elements in the functional mechanism of CF I.

Main Methods:

• UV cross-linking assays to identify RNA-protein interactions.
• In vitro RNA processing assays using wild-type and mutant components.
• In vivo studies using yeast genetics to assess the lethality and functional defects of mutations.

Main Results:

• Rna15 alone does not recognize a specific RNA sequence, but in complex with Hrp1 and Rna14, it specifically binds the A-rich element.
• The interaction of Rna15 with the A-rich element is dependent on Hrp1 binding to the UA-rich motif, mediated by Rna14.
• Mutations in Rna15 affecting RNA binding affinity or in the A-rich element itself disrupt in vitro processing and lead to lethality in vivo, confirming the interaction's importance.

Conclusions:

• The yeast mRNA 3' end processing mechanism involves a novel tethering mechanism where Rna15 is positioned by Rna14 and Hrp1, differing from mammalian systems.
• This specific recognition of RNA elements by protein complexes is essential for the precise definition and maturation of mRNA 3' ends.
• The findings highlight the intricate protein-RNA interactions governing gene expression regulation in eukaryotes.