Polyadenylation Complex Research Articles

3′ adenylation determines mRNA abundance and monitors completion of RNA editing in T. brucei mitochondria

Expression of the mitochondrial genome in protozoan parasite Trypanosoma brucei is controlled post-transcriptionally and requires extensive U-insertion/deletion mRNA editing. In mitochondrial extracts, 3' adenylation reportedly influences degradation kinetics of synthetic edited and pre-edited mRNAs. We have identified and characterized a mitochondrial poly(A) polymerase, termed KPAP1, and determined major polypeptides in the polyadenylation complex. Inhibition of KPAP1 expression abrogates short and long A-tails typically found in mitochondrial mRNAs, and decreases the abundance of never-edited and edited transcripts. Pre-edited mRNAs are not destabilized by the lack of 3' adenylation, whereas short A-tails are required and sufficient to maintain the steady-state levels of partially edited, fully edited, and never-edited mRNAs. The editing directed by a single guide RNA is sufficient to impose a requirement for the short A-tail in edited molecules. Upon completion of the editing process, the short A-tails are extended as (A/U) heteropolymers into structures previously thought to be long poly(A) tails. These data provide the first direct evidence of functional interactions between 3' processing and editing of mitochondrial mRNAs in trypanosomes.

Kinases in the Polyadenylation Complex

After transcription of a gene, the abundance of a protein in a cell can be controlled through various mechanisms operating on the mRNA, including regulation of alternative splicing, polyadenylation, stability and degradation, nuclear export, and accessibility to translation factors. Star-PAP is a noncanonical poly(A) polymerase that has a proline-rich region (PRR), which splits the catalytic domain. Star-PAP functions as part of a larger macromolecular complex. However, in addition to the common proteins found in other polyadenylation complexes, the Star-PAP complex includes phosphatidylinositol-4-phosphate 5 kinase Iα (PIPKIα), and Star-PAP activity is stimulated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. Gonzales et al . showed that the Star-PAP complex isolated from transfected cells also includes protein kinase activity and that Star-PAP is phosphorylated by the associated kinase. The protein kinase activity of the Star-PAP complex was inhibited by the addition of PI(4,5)P 2 , which led the authors to identify the kinase as casein kinase Iα (CKIα), which is known to be inhibited by PI(4,5)P 2 . CKIα and Star-PAP colocalized in nuclear speckles, which are a site of pre-mRNA processing, and inhibitors of CKIα blocked Star-PAP phosphorylation in vitro. Star-PAP is responsible for increasing the abundance of many mRNAs based on microarray experiments; however, CKIα and PIPKIα appear to control the targeting of Star-PAP to a subset of mRNAs involved in oxidative stress. Cells in which CKIα or PIPKIα was decreased using siRNA showed a specific decrease in the abundance of a subset of mRNAs processed by Star-PAP, including two that encode enzymes involved in the response to oxidative stress (HO-1 and NQO1), without affecting other transcripts targeted by Star-PAP. Thus, the presence of a lipid kinase and a protein kinase in the Star-PAP complex appears to target Star-PAP to specific mRNA targets and connects nuclear lipid signaling to mRNA processing. M. L. Gonzales, D. L. Mellman, R. A. Anderson, CKIα is associated with and phosphorylates Star-PAP and is also required for expression of select Star-PAP target messenger RNAs. J. Biol. Chem. 283 , 12665-12673 (2008). [Abstract] [Full Text]

The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3' processing pathway.

Microarray analyses of mRNAs over-expressed in strains lacking the nuclear exosome component Rrp6 identified the transcript encoding the ARE-binding protein Cth2, which functions in cytoplasmic mRNA stability. Subsequent northern analyses revealed that exosome mutants accumulate a 3′-extended transcript at the expense of the mature CTH2 mRNA. The 3′ ends of the CTH2 mRNA were mapped to a [GU3–5]5 repeat, unlike any previously characterized polyadenylation site. CTH2 mRNA accumulation was not inhibited by mutations in 3′-cleavage and polyadenylation factors, Rna14, Rna15 and Pap1, which block accumulation of other mRNAs. The 3′-extended CTH2 pre-mRNA strongly accumulated in strains with mutations in the TRAMP4 polyadenylation complex or the Nrd1/Nab3/Sen1 complex, and contains multiple Nrd1 and Nab3 binding sites. CTH2 carries a consensus ARE element and levels of the pre-mRNA and mRNA were elevated by mutation of the ARE or inactivation of the nuclear 5′-exonuclease Rat1. We propose that CTH2 mRNA is processed from a 3′-extended primary transcript by the exosome, TRAMP and Nrd1/Nab3/Sen1 complexes. This unusual pathway may allow time for nuclear, ARE-mediated regulation of CTH2 levels involving Rat1.

Redox and heavy metal effects on the biochemical activities of an Arabidopsis polyadenylation factor subunit

The Arabidopsis CPSF30 ortholog (AtCPSF30) is an RNA-binding endonuclease that is part of the plant polyadenylation complex. Previous work (B. Addepalli, A.G. Hunt, Nucleic Acids Res. 35 (2007) 4453–4463) demonstrated that different zinc finger motifs in the protein were responsible for RNA-binding and nuclease activity, respectively. In this study, a more detailed functional map of AtCPSF30 is presented, a map that includes descriptions of novel biochemical activities. Elevated temperatures, the specific zinc chelator 1,10-phenanthroline, and the sulfhydryl reagent dithiothreitol all had differential inhibitory effects on the RNA-binding and nuclease activities. The endonuclease activity of AtCPSF30 was inhibited by relatively high (>100μM) concentrations of zinc, and this inhibition required a plant-specific N-terminal domain apart from the zinc finger core of the protein. ATP stimulated the nuclease activity in the presence of zinc, and this stimulation required a plant-specific C-terminal domain, again apart from the zinc finger core. These studies reveal a subtle and unexpected complexity to AtCPSF30, and raise the possibility that multiple avenues of regulation may impinge on this protein through different functional domains.

The nuclear RNA surveillance machinery: The link between ncRNAs and genome structure in budding yeast?

The TRAMP polyadenylation complexes have well-established functions in RNA surveillance, stimulating degradation by the 3' to 5' exonuclease activity of the exosome on a wide range of nuclear RNAs and RNA-protein complexes. Known targets include some of the non-protein coding RNA transcripts (ncRNAs), which are apparently widely transcribed from yeast and mammalian genomes. We will discuss potential mechanisms of TRAMP recruitment and exosome activation during RNA surveillance and degradation. Less well-understood observations link both the TRAMP and exosome complexes to chromatin structure and DNA repair, and we will speculate on the potential significance of these activities.

Xenopus Rbm9 is a novel interactor of XGld2 in the cytoplasmic polyadenylation complex

During early development, control of the poly(A) tail length by cytoplasmic polyadenylation is critical for the regulation of specific mRNA expression. Gld2, an atypical poly(A) polymerase, is involved in cytoplasmic polyadenylation in Xenopus oocytes. In this study, a new XGld2-interacting protein was identified: Xenopus RNA-binding motif protein 9 (XRbm9). This RNA-binding protein is exclusively expressed in the cytoplasm of Xenopus oocytes and interacts directly with XGld2. It is shown that XRbm9 belongs to the cytoplasmic polyadenylation complex, together with cytoplasmic polyadenylation element-binding protein (CPEB), cleavage and polyadenylation specificity factor (CPSF) and XGld2. In addition, tethered XRbm9 stimulates the translation of a reporter mRNA. The function of XGld2 in stage VI oocytes was also analysed. The injection of XGld2 antibody into oocytes inhibited polyadenylation, showing that endogenous XGld2 is required for cytoplasmic polyadenylation. Unexpectedly, XGld2 and CPEB antibody injections also led to an acceleration of meiotic maturation, suggesting that XGld2 is part of a masking complex with CPEB and is associated with repressed mRNAs in oocytes.

The Interaction Between Two Arabidopsis Polyadenylation Factor Subunits Involves an Evolutionarily-Conserved Motif and Has Implications for the Assembly and Function of the Polyadenylation Complex

The polyadenylation factor subunit "Factor Interacting with Poly(A) polymerase" (Fip1) is an important bridging subunit in the eukaryotic polyadenylation complex. To better understand the functioning of Fip1 in Arabidopsis, a random combinatorial screen for peptides that interact with a conserved plant-specific domain in the protein was conducted. A search of the Arabidopsis proteome using these Fip1-binding peptides as queries resulted in the identification of a number of putative Fip1-interacting proteins. One of these was the polyadenylation factor subunit, CstF77. This purported interaction was confirmed by yeast two-hybrid and in vitro assays. Mutation of the motif identified in the phage display screen eliminated the interaction, corroborating the results of the phage display screen. The domain of CstF77 that interacts with Fip1 lies at its extreme C-terminus and is distinct from the part of CstF77 that binds CstF64. Taken together, these results suggest that Fip1 is situated near CstF64 in the polyadenylation complex.

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

BackgroundThe polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis.ResultsBy protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17%) showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations.ConclusionAn extensive protein network was revealed for plant polyadenylation machinery, in which all predicted proteins were found to be connecting to the complex. The gene expression profiles are indicative that specialized sub-complexes may be formed to carry out targeted processing of mRNA in different developmental stages and tissue types. These results offer a roadmap for further functional characterizations of the protein factors, and for building models when testing the genetic contributions of these genes in plant growth and development.

Sumoylation Modulates the Assembly and Activity of the Pre-mRNA 3′ Processing Complex

Eukaryotic pre-mRNA 3'-end formation is catalyzed by a complex set of factors that must be intricately regulated. In this study, we have discovered a novel role for the small ubiquitin-like modifier SUMO in the regulation of mammalian 3'-end processing. We identified symplekin, a factor involved in complex assembly, and CPSF-73, an endonuclease, as SUMO modification substrates. The major sites of sumoylation in symplekin and CPSF-73 were determined and found to be highly conserved across species. A sumoylation-deficient mutant was defective in rescuing cell viability in symplekin small interfering RNA (siRNA)-treated cells, supporting the importance of this modification in symplekin function. We also analyzed the involvement of sumoylation in 3'-end processing by altering the sumoylation status of nuclear extracts. This was done by the addition of a SUMO protease, which we show interacts with both symplekin and CPSF-73, or by siRNA-mediated depletion of ubc9, the SUMO E2-conjugating enzyme. Both treatments resulted in a marked inhibition of processing. The assembly of a functional polyadenylation complex was also impaired by the SUMO protease. Our identification of two key polyadenylation factors as SUMO targets and of the role of SUMO in enhancing the assembly and activity of the 3'-end-processing complex together reveal an important function for SUMO in the processing of mRNA precursors.

RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

One activity that controls mRNA translation in vertebrate oocytes, embryos, and neurons is cytoplasmic polyadenylation. In Xenopus oocytes, where much of the biochemistry of this process has been elucidated, nuclear pre-mRNAs containing a cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) have long poly(A) tails; once the RNAs are spliced and transported to the cytoplasm, the tails are shortened. Following the resumption of meiosis, the poly(A) tails are lengthened and translation ensues. CPEB is a sequence-specific RNA-binding protein that coordinates these events and does so by binding to the CPE as well as several factors including Gld2, a poly(A) polymerase, and PARN [poly(A)-specific ribonuclease], a deadenylase. Here, we show that ePAB, embryonic poly(A)-binding protein, transiently associates with the polyadenylation complex; it initially interacts with CPEB, but after polyadenylation, it binds the poly(A) tail. ePAB dissociation from CPEB is regulated by RINGO (Rapid Inducer of G(2)/M progression in Oocytes), a cyclin B1-like cofactor that activates cdk1, a protein kinase that phosphorylates CPEB. Subsequent ePAB binding to the poly(A) tail is necessary to protect the homopolymer from degradation by deadenylating enzymes. Poly(A)-bound ePAB also interacts with eIF4G, which instigates translation initiation of CPEB-bound mRNAs.

The Exosome Subunit Rrp44 Plays a Direct Role in RNA Substrate Recognition

The exosome plays key roles in RNA maturation and surveillance, but it is unclear how target RNAs are identified. We report the functional characterization of the yeast exosome component Rrp44, a member of the RNase II family. Recombinant Rrp44 and the purified TRAMP polyadenylation complex each specifically recognized tRNA(i)(Met) lacking a single m(1)A(58) modification, even in the presence of a large excess of total tRNA. This tRNA is otherwise mature and functional in translation in vivo but is presumably subtly misfolded. Complete degradation of the hypomodified tRNA required both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 failed to degrade tRNA(i)(Met), showing this to be a specific Rrp44 substrate. Recognition of hypomodified tRNA(i)(Met) by Rrp44 is genetically separable from its catalytic activity on other substrates, with the mutations mapping to distinct regions of the protein.

Opposing Polymerase-Deadenylase Activities Regulate Cytoplasmic Polyadenylation

Cytoplasmic polyadenylation is one mechanism that regulates translation in early animal development. In Xenopus oocytes, polyadenylation of dormant mRNAs, including cyclin B1, is controlled by the cis-acting cytoplasmic polyadenylation element (CPE) and hexanucleotide AAUAAA through associations with CPEB and CPSF, respectively. Previously, we demonstrated that the scaffold protein symplekin contacts CPEB and CPSF and helps them interact with Gld2, a poly(A) polymerase. Here, we report the mechanism by which poly(A) tail length is regulated. Cyclin B1 pre-mRNA acquires a long poly(A) tail in the nucleus that is subsequently shortened in the cytoplasm. The shortening is controlled by CPEB and PARN, a poly(A)-specific ribonuclease. Gld2 and PARN both reside in the CPEB-containing complex. However, because PARN is more active than Gld2, the poly(A) tail is short. When oocytes mature, CPEB phosphorylation causes PARN to be expelled from the ribonucleoprotein complex, which allows Gld2 to elongate poly(A) by default.

Cytoplasmic CstF-77 Protein Belongs to a Masking Complex with Cytoplasmic Polyadenylation Element-binding Protein in Xenopus Oocytes

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

Surveillance of nuclear-restricted pre-ribosomes within a subnucleolar region of Saccharomyces cerevisiae.

We previously hypothesized that HEAT-repeat (Huntington, elongation A subunit, TOR) ribosome synthesis factors function in ribosome export. We report that the HEAT-repeat protein Sda1p is a component of late 60S pre-ribosomes and is required for nuclear export of both ribosomal subunits. In strains carrying the ts-lethal sda1-2 mutation, pre-60S particles were rapidly degraded following transfer to 37 degrees C. Polyadenylated forms of the 27S pre-rRNA and the 25S rRNA were detected, suggesting the involvement of the Trf4p/Air/Mtr4p polyadenylation complex (TRAMP). The absence of Trf4p suppressed polyadenylation and stabilized the pre-rRNA and rRNA. The absence of the nuclear exosome component Rrp6p also conferred RNA stabilization, with some hyperadenylation. We conclude that the nuclear-restricted pre-ribosomes are polyadenylated by TRAMP and degraded by the exosome. In sda1-2 strains at 37 degrees C, pre-40S and pre-60S ribosomes initially accumulated in the nucleoplasm, but then strongly concentrated in a subnucleolar focus, together with exosome and TRAMP components. Localization of pre-ribosomes to this focus was lost in sda1-2 strains lacking Trf4p or Rrp6p. We designate this nucleolar focus the No-body and propose that it represents a site of pre-ribosome surveillance.

Yeast Trf5p is a nuclear poly(A) polymerase

Recent analyses have shown that the activity of the yeast nuclear exosome is stimulated by the Trf4p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex. Here, we report that strains lacking the Rrp6p component of the nuclear exosome accumulate polyadenylated forms of many different ribosomal RNA precursors (pre-rRNAs). This polyadenylation is reduced in strains lacking either the poly(A) polymerase Trf4p or its close homologue Trf5p. In contrast, polyadenylation is enhanced by overexpression of Trf5p. Polyadenylation is also markedly increased in strains lacking the RNA helicase Mtr4p, indicating that it is required to couple poly(A) polymerase activity to degradation. Tandem affinity purification-tagged purified Trf5p showed polyadenylation activity in vitro, which was abolished by a double point mutation in the predicted catalytic site. Trf5p co-purified with Mtr4p and Air1p, indicating that it forms a complex, designated TRAMP5, that has functions that partially overlap with the TRAMP complex.

An Arabidopsis Fip1 Homolog Interacts with RNA and Provides Conceptual Links with a Number of Other Polyadenylation Factor Subunits

The protein Fip1 is an important subunit of the eukaryotic polyadenylation apparatus, since it provides a bridge of sorts between poly(A) polymerase, other subunits of the polyadenylation apparatus, and the substrate RNA. In this study, a previously unreported Arabidopsis Fip1 homolog is characterized. The gene for this protein resides on chromosome V and encodes a 1196-amino acid polypeptide. Yeast two-hybrid and in vitro assays indicate that the N-terminal 137 amino acids of the Arabidopsis Fip1 protein interact with poly(A) polymerase (PAP). This domain also stimulates the activity of the PAP. Interestingly, this part of the Arabidopsis Fip1 interacts with Arabidopsis homologs of CstF77, CPSF30, CFIm-25, and PabN1. The interactions with CstF77, CPSF30, and CFIm-25 are reminiscent in various respects of similar interactions seen in yeast and mammals, although the part of the Arabidopsis Fip1 protein that participates in these interactions has no apparent counterpart in other eukaryotic Fip1 proteins. Interactions between Fip1 and PabN1 have not been reported in other systems; this may represent plant-specific associations. The C-terminal 789 amino acids of the Arabidopsis Fip1 protein were found to contain an RNA-binding domain; this domain correlated with an intact arginine-rich region and had a marked preference for poly(G) among the four homopolymers studied. These results indicate that the Arabidopsis Fip1, like its human counterpart, is an RNA-binding protein. Moreover, they provide conceptual links between PAP and several other Arabidopsis polyadenylation factor subunit homologs.

A nuclear surveillance pathway for mRNAs with defective polyadenylation.

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3'-5' exonucleases, accumulation of poly(A)+ mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6delta. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3'-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation.

Vertebrate GLD2 poly(A) polymerases in the germline and the brain

Cytoplasmic polyadenylation is important in the control of mRNA stability and translation, and for early animal development and synaptic plasticity. Here, we focus on vertebrate poly(A) polymerases that are members of the recently described GLD2 family. We identify and characterize two closely related GLD2 proteins in Xenopus oocytes, and show that they possess PAP activity in vivo and in vitro and that they bind known polyadenylation factors and mRNAs known to receive poly(A) during development. We propose that at least two distinct polyadenylation complexes exist in Xenopus oocytes, one of which contains GLD2; the other, maskin and Pumilio. GLD2 protein interacts with the polyadenylation factor, CPEB, in a conserved manner. mRNAs that encode GLD2 in mammals are expressed in many tissues. In the brain, mouse, and human GLD2 mRNAs are abundant in anatomical regions necessary for long-term cognitive and emotional learning. In the hippocampus, mouse GLD2 mRNA colocalizes with CPEB1 and Pumilio1 mRNAs, both of which are likely involved in synaptic plasticity. We suggest that mammalian GLD2 poly(A) polymerases are important in synaptic translation, and in polyadenylation throughout the soma.

RNA Degradation by the Exosome Is Promoted by a Nuclear Polyadenylation Complex

The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.

Recognition of GU-rich polyadenylation regulatory elements by human CstF-64 protein.

Vertebrate polyadenylation sites are identified by the AAUAAA signal and by GU-rich sequences downstream of the cleavage site. 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. 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. 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 propose that this conformational change initiates assembly. Consecutive Us are required for a strong CstF-GU interaction and we show how UU dinucleotides are recognized. Contacts outside the UU pocket fine tune the protein-RNA interaction and provide different affinities for distinct GU-rich elements. The protein-RNA interface remains mobile, most likely a requirement to bind many GU-rich sequences and yet discriminate against other RNAs. The structural distinction between sequences that form stable and unstable complexes provides an operational distinction between weakly and strongly processed poly(A) sites.