Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition

Abstract

At least half of all human pre-mRNAs are subject to alternative 3′ processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3′ UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3′ processing factor CFI m can function as a primary determinant of poly(A) site recognition in the absence of the A(A/U)UAAA motif. CFI m is sufficient to direct sequence-specific, A(A/U)UAAA-independent poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the RNA substrate. ChIP analysis indicates that CFI m is recruited to the transcription unit, along with CPSF and CstF, during the initial stages of transcription, supporting a direct role for CFI m in poly(A) site recognition. The recognition of three distinct sequence elements by CFI m , CPSF, and CstF suggests that vertebrate poly(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.