Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I

Abstract

The accuracy of the 3′-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of heterodimers with Rna15 to stabilize the RNA-protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14-Rna15 heterodimer in the presence and absence of 3′-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis.mRNA processing | mRNA transport | protein-RNA | methyl-TROSY | modeling E ukaryotic messenger RNA precursors (pre-mRNA) undergo extensive cotranscriptional processing before their transport to the cytoplasm and subsequent translation (1, 2). The processing events include 5′-end capping, splicing to remove intronic sequences and cleavage and polyadenylation at the 3′-end (3, 4). Correct 3′-end processing is necessary for mRNA biogenesis and transcription termination, preserves RNAs from degradation (5), promotes its export to the cytoplasm (6), enhances translation (7) and promotes transcriptional activation (4, 8). Cleavage and polyadenylation can be uncoupled in vitro but in vivo they are connected with each other and with transcription termination. Defects in 3′-end processing have also been associated with several diseases in humans (9), but this process may be even more critical in yeast that has shorter intergenic regions and much rarer alternative splicing (10, 11). In this organism, efficient mRNA transport is mostly dependent on polyadenylation rather than splicing.In yeast, the 3′-end processing reaction is executed by a complex machine containing more than 20 proteins (4) which share significant similarities with human 3′-end processing proteins. The entire yeast 3′-end processing complex is divided into two subcomplexes, called cleavage factor I (CF I) and cleavage and polyadenylation factor (CPF) (4). CF I binds to the pre-mRNA upstream of the cleavage signal within the pre-mRNA, whereas CPF binds at the cleavage site as well as upstream and downstream of the cleavage site. Several sequences within yeast pre-mRNAs, which are surprisingly poorly conserved, direct the recruitment of cleavage and polyadenylation factors (12, 13). In the 5′-3′ direction, they include AU-rich efficiency element (EE) responsible for polyadenylation efficiency; A-rich positioning element (PE) critical for precise 3′-end processing; t...

Distinct roles of Pcf11 zinc-binding domains in pre-mRNA 3′-end processing

“…Subunit–subunit interactions in CF IA have been reported through pairwise assays either with the two-hybrid analysis or by pairwise association/expression ( 13 , 16 , 17 , 20 , 41 , 43 , 53 ) giving rise to a refined interaction map in CF IA ( 26 ). However, interactions between two proteins that require a third stabilizing protein may not be detected, as for example with the PYM protein interaction with Mago and Y14 ( 54 ).…”

“…Molecular characterization of the RNA recognition motif (RRM) domain of Rna15, and the orthologue CstF-64, reveal aspects of pre-mRNA binding ( 23 ). Increased RNA sequence specificity has also been demonstrated with structural details of the auxiliary subunit Hrp1 (also known as CF IB or Nab4), and in particular within a Hrp1–Rna15–RNA ternary complex ( 24 – 26 ). A connection to the largest subunit of RNA polymerase II, Rpb1 is mediated by the C-terminal interacting domain (CID) of Pcf11 that binds to the Rpb1 C-terminal YSPTSPS heptamer repeats with phosphorylation on the serine in position 2 ( 27 , 28 ).…”

RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination

“…Like Hrp1, several of these proteins may contribute to DEF1 pA 1 site recognition. Rna15 and Rna14 are also members of CFI, and while Rna15 recognizes pA site positioning elements, Rna14 is capable of bridging Rna15 and Hrp1 in a CFI complex ( Gross and Moore 2001 ; Barnwal et al 2012 ). Cft2 is a component of the core CPF that binds the CYC1 pA site in vitro and crosslinks near pA sites in vivo , as well as interacting with the Pol II CTD ( Dichtl and Keller 2001 ; Kyburz et al 2003 ; Baejen et al 2014 ).…”

Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery

“…It has been suggested that Rna15, when complexed with Hrp1, utilizes the secondary site to recognize the A-rich PE (99) (Fig. 2F) and by itself recognizes a U-rich consensus upstream of the cleavage site (26, 27) in a scenario in which CFI contains two copies of Rna15 but only one copy of Hrp1 (88,100).…”