Creatine Phosphate, Not ATP, Is Required for 3′ End Cleavage of Mammalian Pre-mRNA in Vitro

Yutaka Hirose,James L Manley

doi:10.1074/jbc.272.47.29636

Abstract

The poly(A) tail of a mammalian mRNA is generated by endonucleolytic cleavage and poly(A) addition. Previous studies conducted with nuclear extracts suggested an ATP requirement for the cleavage step. We have reexamined the cofactor requirement, initially with the SV40 late pre-mRNA, which requires for cleavage four protein factors, cleavage and polyadenylation specificity factor, cleavage stimulation factor, cleavage factor I, and cleavage factor II. Using highly purified preparations of these factors, which lacked detectable creatine phosphokinase and ATPase activities, creatine phosphate (CP) was, surprisingly, found to be sufficient to promote efficient cleavage. Although other phosphate compounds substituted poorly or not at all for CP, another phosphoguanidine, arginine phosphate, was fully functional. Notably, ATP was neither necessary nor sufficient, and could in fact inhibit the reaction. Treatment of the purified factors with hexokinase plus glucose (to deplete any contaminating ATP) was without effect, as was addition of EDTA. Using 32P-labeled CP, we found that neither hydrolysis of CP nor phosphate transfer from CP occurred during the cleavage reaction. CP also allowed cleavage of the adenovirus 2 L3 pre-mRNA. However, in this case, ATP both enhanced the reaction and influenced the precise site of cleavage, perhaps reflecting the requirement of poly(A) polymerase for cleavage of this RNA. These results indicate that ATP is not essential for 3' pre-mRNA cleavage and that CP or a related compound can function as a necessary cofactor.

Highlights

All eukaryotic mRNAs, with the exception of major histone mRNAs, have a poly(A) tail at their 3Ј end
The process generating the poly(A) tail, referred to as 3Ј end formation, involves endonucleolytic cleavage of the precursor RNA followed by the sequential addition of adenylate residues. 3Ј end formation is an essential step in the maturation of pre-mRNAs, and it appears to be coupled with other events in the nucleus, including transcription termination [1,2,3] and mRNA splicing (4 – 6)
Creatine Phosphate Is Required for Efficient Cleavage—Our previous experiments using a relatively crude preparation of 3Ј processing factors demonstrated that efficient cleavage could be detected in the presence of creatine phosphate (CP) without added ATP, whereas addition of ATP alone did not allow cleavage [15]

Summary

Introduction

All eukaryotic mRNAs, with the exception of major histone mRNAs, have a poly(A) tail at their 3Ј end. A standard 12.5-␮l cleavage reaction mixture contained 0.25 ␮l of CstF; 2.0 ␮l of CPSF; 1.75 ␮l of CFI; 2.0 ␮l of CFII; 0.25 ␮l of either Buffer D (for SV40 pre-mRNA) or recombinant bovine PAP (for L3 pre-mRNA); 10 mM HEPES-NaOH, pH 7.9; 10% glycerol; 25 mM (NH4)2SO4; 0.1 mM EDTA; 0.25 mM dithiothreitol; 0.25 mM phenylmethylsulfonyl fluoride; 2.5% polyvinyl alcohol; 0.2– 0.5 ng of substrate RNA; 500 ng of E. coli tRNA; and 2 mM EDTA, 0.5 mM MgCl2, or no divalent cation as indicated, plus the indicated amounts of CP, ATP, or other potential cofactors.

Results

Conclusion