Despite its obvious importance, structural and mechanistic insight on how alternative mRNA processing decisions are executed remains sparse. More puzzling are situations where poly(A) site selection is controlled instead by altering the intracellular levels of general processing factors such as CstF-64 ( Takagaki et al., 1996 Colgan and Manley, 1997 Takagaki and Manley, 1998). ![]() ![]() In some cases, of which U1A is the best understood example ( Boelens et al., 1993), the timing and efficiency of polyadenylation decisions are controlled by gene-specific factors and their interaction with specific RNA elements and with the polyadenylation machinery. In polyadenylation, as in splicing, the efficiency with which different processing sites are utilized is controlled by regulatory cis-acting RNA elements and their interactions with trans-acting protein factors. Differential usage of alternative poly(A) sites produces mature transcripts with distinctive 3′-UTRs and gene expression profiles and, sometimes, even different coding sequences. Furthermore, an increasing number of developmental and differentiation decisions are now known to be executed by alternative polyadenylation of the same mRNA ( Barabino and Keller, 1999 Zhao et al., 1999). However, ∼30% of all human mRNAs contain alternative polyadenylation signals ( Beaudoing et al., 2000). Alternative splicing has long been recognized as a major source of diversity in gene expression, while polyadenylation has been considered largely constitutive. Maturation of mRNA precursors is a constitutive process for most transcripts but is also a major regulatory event in eukaryotic cells, producing multiple isoforms of the same mRNA by alternative processing. The structural distinction between sequences that form stable and unstable complexes provides an operational distinction between weakly and strongly processed poly(A) sites. The protein–RNA interface remains mobile, most likely a requirement to bind many GU-rich sequences and yet discriminate against other RNAs. Contacts outside the UU pocket fine tune the protein–RNA interaction and provide different affinities for distinct GU-rich elements. Consecutive Us are required for a strong CstF–GU interaction and we show how UU dinucleotides are recognized. We propose that this conformational change initiates assembly. The C-terminal helix unfolds upon RNA binding and extends into the hinge domain where interactions with factors responsible for assembly of the polyadenylation complex occur. We present the structure of the RNA-binding domain of CstF-64 containing an RNA recognition motif (RRM) augmented by N- and C-terminal helices. These are recognized by a heterotrimeric protein complex (CstF) through its 64 kDa subunit (CstF-64) the strength of this interaction affects the efficiency of poly(A) site utilization. Vertebrate polyadenylation sites are identified by the AAUAAA signal and by GU-rich sequences downstream of the cleavage site.
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