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

The 3′ ends of most eukaryotic mRNAs undergo maturation before export of the transcript to the cytoplasm. This maturation occurs in two steps that are tightly coupled in vivo but can be experimentally uncoupled in vitro. First, the nascent transcript is cleaved at a specific site downstream of the translational stop codon, and then a poly(A) tail is added. Processing is performed by a multisubunit complex that provides the nuclease activity and also confers sequence specificity to a template-independent poly(A) polymerase (PAP) (43, 68, 71). This event regulates the efficiency of transcriptional termination (14, 29, 51), nuclear export (13, 30, 36), and translation (54) and contributes to the longevity of the RNA in the cytoplasm (46, 63, 70). The choice of poly(A) site can even change the amount of sequence found in the mRNA and is thus an additional mechanism for the control of gene expression (18). An intricate dance involving the recognition of cis-acting signals in the pre-mRNA by multisubunit protein factors defines the cleavage site by directing the processing machinery to the appropriate location on the nascent transcript (25, 71). In mammals, cleavage of the nascent mRNA precursor occurs at a loosely conserved element called the poly(A) site, usually containing a CA dinucleotide. This site is flanked by three other sequences which control the exact location and efficiency of cleavage. These are called the A-rich element, the downstream element, and the upstream element. The A-rich element, most often the sequence AAUAAA, is 10 to 30 nucleotides upstream from the poly(A) site. This element is highly conserved within the animal, plant, and fungal kingdoms. The downstream element is U-rich or GU-rich and lies within 50 nucleotides downstream of the poly(A) site. Mutations in either element affect the efficiency of the 3′-end processing machinery, and the positioning of the cleavage site is affected by the distance between these two signals. Upstream elements, found in some viral and cellular genes, are not essential but stimulate the overall efficiency of processing. Four multisubunit protein factors, plus RNA polymerase II, are required for cleavage of the mammalian mRNA precursor and have been extensively characterized biochemically. They are called cleavage-polyadenylation specificity factor (CPSF), cleavage stimulatory factor (CstF), and mammalian cleavage factors I and II (CF IM and CF IIM). Cleavage is most efficient in the presence of PAP and RNA polymerase II. Polyadenylation of the cleaved transcript requires CPSF, poly(A) binding protein II (PAB II), and PAP (43, 68, 71). The actual 3′-end processing reaction occurs within a structure created through interactions of these factors with each other and with the pre-mRNA. The binding of CPSF to the A-rich sequence and of CstF to the downstream element is strengthened through protein-mediated interactions of the two factors (43, 71). Since CF IM and CF IIM are required for the cleavage step, these factors may be directed to the poly(A) site through interactions with bound CPSF or CstF (15) and through the binding of CF IM to the mRNA (52). The interaction of CPSF at the AAUAAA signal also gives specificity to the PAP, and the binding of PAB II to the nascent poly(A) tail stabilizes the polyadenylation complex (7). In Saccharomyces cerevisiae a similar but not identical machinery is involved in 3′ end maturation. At least four different types of elements contribute to the efficiency and accuracy of yeast 3′-end processing (26). As in mammals, the poly(A) site is the location of endonucleolytic cleavage of the mRNA precursor, which then becomes the site of initiation for polymerization of the adenosine tail. Mutagenesis studies with the CYC1 and ADH1 genes (27, 53) have defined the poly(A) site as Py(A)n. As in mammals, the A-rich sequence, ca. 20 bases upstream of the cleavage site, is extremely well conserved (22, 66). In vivo, mutations in this element most often do not affect the overall efficiency of processing but instead cause a defocusing of the endonucleolytic activity through the activation of previously cryptic poly(A) sites. Because of this effect, this motif has often been referred to as the positioning element (25). Two global sequence analyses detected a bias toward U-rich sequences immediately upstream and downstream of the poly(A) site (22, 66), and mutation of these sequences affects 3′-end formation in vitro and in vivo (16). However, additional downstream sequence does not appear to be very important for in vivo processing (3, 55), a finding consistent with the finding that precursors with only 7 to 10 nucleotides beyond the cleavage site are readily processed in vitro (10, 11). Instead of a downstream element, yeast mRNAs contain a functionally analogous sequence which is UA-rich in nature and located upstream of the A-rich sequence. This sequence can function from up to 30 nucleotides away from the A-rich sequence and, in some genes, in both orientations. Mutations in this sequence reduce the overall efficiency but not the accuracy of the cleavage reaction on the mRNA in vivo (71). The machinery required for processing of yeast mRNA 3′ ends consists of four factors, defined by chromatographic fractionation of whole-cell extract (11). CF I and II recognize the processing signals on the mRNA and perform the endonucleolytic cleavage, whereas CF I, polyadenylation factor I (PF I), and PAP are required for the polyadenylation step. A factor containing the CF II subunits combined with a cohort of polyadenylation-specific proteins can be isolated by affinity chromatography (50). This complex can provide both CF II and PF I activities and has been designated cleavage-polyadenylation factor (CPF). Unlike the mammalian system, much less is known about the means through which these factors recognize the cis elements contained in the pre-mRNA. When purified CF II is used in cross-linking experiments, the Cft2 subunit cross-links to a model mRNA precursor only if the substrate contains the UA-rich element and sequence beyond the poly(A) site (72). A recent study using RNase H protection analysis has found that CPF, as well as recombinant Cft2, interacts with the region around the poly(A) site (16). CF I contains two proteins with obvious RNP-type RNA-binding motifs: Rna15 (45) and Hrp1 (33). Hrp1 has been shown to require the UA-rich element for binding to mRNA (12, 33), and SELEX experiments with Hrp1 have selected (UA)3 sequences (65). Mutations in the RNA binding domains of Hrp1 generate a thermosensitive phenotype (33), implying that recognition of the UA-rich element is an essential step in mRNA processing. Rna15 contains a single RNA recognition motif (RRM) domain, but its binding has not been mapped to a particular site on the mRNA. In vivo, yeast carrying a thermosensitive mutation at the RNA15 locus display defocused polyadenylation (17), mimicking mutations at the A-rich element and implying a role for this protein in contributing to the specificity of the cleavage reaction. In the study presented here, we use CF I reconstituted from separately expressed and purified proteins (23) to directly demonstrate the binding of Rna15 to the A-rich element. We show that Rna15 in the absence of other CF I proteins does not recognize a specific RNA sequence but gains this ability when assembled into a subcomplex of CF I containing Hrp1 and Rna14, a protein previously shown to bridge Rna15 and Hrp1 (23). Furthermore, through an Rna15 construct with attenuated RNA binding, we show that Rna15 recognition of the A-rich element is an essential step in mRNA 3′-end formation. Our identification of the RNA sequences recognized by CF I illustrates a central but previously unknown aspect of the mechanism by which the yeast 3′ end is defined and processed.