Poly(A) tail length control is caused by termination of processive synthesis

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Metabolism

Background:

• Polyadenylation is a crucial post-transcriptional modification affecting mRNA stability and translation.
• Poly(A) polymerase (PAP) synthesizes the poly(A) tail, but its regulation is complex.
• Specific protein factors, including CPSF and PAB II, are known to influence PAP activity.

Purpose of the Study:

• To elucidate the mechanism by which poly(A) polymerase controls poly(A) tail length.
• To investigate the roles of CPSF and PAB II in poly(A) tail length determination.
• To understand how rapid and processive synthesis transitions to slower elongation.

Main Methods:

• In vitro assays using purified proteins and synthetic poly(A) RNA substrates.
• Analysis of poly(A) polymerase activity with varying poly(A) tail lengths.
• Characterization of protein-RNA interactions and their influence on enzyme kinetics.

Main Results:

• Poly(A) polymerase synthesizes poly(A) tails processively only when bound by both CPSF and PAB II.
• A burst of synthesis yields tails of approximately 250 nucleotides, after which elongation slows.
• Poly(A) tail length is measured by the number of PAB II molecules bound, which dictates the switch from processive to distributive elongation.

Conclusions:

• Poly(A) tail length is precisely controlled by protein-RNA interactions, specifically the binding of PAB II.
• The transition from rapid to slow elongation is mediated by the inability of CPSF and PAB II to simultaneously stimulate PAP on longer tails.
• This mechanism ensures accurate poly(A) tail length, independent of ATP hydrolysis or nucleotide turnover.