Structural Insights into the Human Pre-mRNA 3'-End Processing Machinery.

The mammalian pre-mRNA 3'-end-processing machinery consists of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and other proteins, but the overall architecture of this machinery remains unclear. CPSF contains two functionally distinct modules: a cleavage factor (mCF) and a polyadenylation specificity factor (mPSF). Here, we have produced recombinant human CPSF and CstF and examined these factors by electron microscopy (EM). We find that mPSF is the organizational core of the machinery, while the conformations of mCF and CstF and the position of mCF relative to mPSF are highly variable. We have identified by cryo-EM a segment in CPSF100 that tethers mCF to mPSF, and we have named it the PSF interaction motif (PIM). Mutations in the PIM can abolish CPSF formation, indicating that it is a crucial contact in CPSF. We have also obtained reconstructions of mCF and CstF77 by cryo-EM, assembled around the mPSF core.

Human CSTF2 RNA Recognition Motif Domain Binds to a U-Rich RNA Sequence through a Multistep Binding Process.

The RNA recognition motif (RRM) is a conserved and ubiquitous RNA-binding domain that plays essential roles in mRNA splicing, polyadenylation, transport, and stability. RRM domains exhibit remarkable diversity in binding partners, interacting with various sequences of single- and double-stranded RNA, despite their small size and compact fold. During pre-mRNA cleavage and polyadenylation, the RRM domain from CSTF2 recognizes U- or G/U-rich RNA sequences downstream from the cleavage and polyadenylation site to regulate the process. Given the importance of alternative cleavage and polyadenylation in increasing the diversity of mRNAs, the exact mechanism of binding of RNA to the RRM of CSTF2 remains unclear, particularly in the absence of a structure of this RRM bound to a native RNA substrate. Here, we performed a series of NMR titration and spin relaxation experiments, which were complemented by paramagnetic relaxation enhancement measurements and rigid-body docking, to characterize the interactions of the CSTF2 RRM with a U-rich ligand. Our results reveal a multistep binding process involving differences in ps-ns time scale dynamics and potential structural changes, particularly in the C-terminalα-helix. These results provide insights into how the CSTF2 RRM domain binds to U-rich RNA ligands and offer a greater understanding for the molecular basis of the regulation of pre-mRNA cleavage and polyadenylation.

A missense mutation in the CSTF2 gene that impairs the function of the RNA recognition motif and causes defects in 3' end processing is associated with intellectual disability in humans.

CSTF2 encodes an RNA-binding protein that is essential for mRNA cleavage and polyadenylation (C/P). No disease-associated mutations have been described for this gene. Here, we report a mutation in the RNA recognition motif (RRM) of CSTF2 that changes an aspartic acid at position 50 to alanine (p.D50A), resulting in intellectual disability in male patients. In mice, this mutation was sufficient to alter polyadenylation sites in over 1300 genes critical for brain development. Using a reporter gene assay, we demonstrated that C/P efficiency of CSTF2D50A was lower than wild type. To account for this, we determined that p.D50A changed locations of amino acid side chains altering RNA binding sites in the RRM. The changes modified the electrostatic potential of the RRM leading to a greater affinity for RNA. These results highlight the significance of 3' end mRNA processing in expression of genes important for brain plasticity and neuronal development.

The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity.

Cleavage and polyadenylation (C/P) of mRNA is an important cellular process that promotes increased diversity of mRNA isoforms and could change their stability in different cell types. The cleavage stimulation factor (CstF) complex, part of the C/P machinery, binds to U- and GU-rich sequences located downstream from the cleavage site through its RNA-binding subunit, CstF-64. Less is known about the function of the other two subunits of CstF, CstF-77 and CstF-50. Here, we show that the carboxy-terminus of CstF-77 plays a previously unrecognized role in enhancing C/P by altering how the RNA recognition motif (RRM) of CstF-64 binds RNA. In support of this finding, we also show that CstF-64 relies on CstF-77 to be transported to the nucleus; excess CstF-64 localizes to the cytoplasm, possibly via interaction with cytoplasmic RNAs. Reverse genetics and nuclear magnetic resonance studies of recombinant CstF-64 (RRM-Hinge) and CstF-77 (monkeytail-carboxy-terminal domain) indicate that the last 30 amino acids of CstF-77 increases the stability of the RRM, thus altering the affinity of the complex for RNA. These results provide new insights into the mechanism by which CstF regulates the location of the RNA cleavage site during C/P.

Reconstitution of the CstF complex unveils a regulatory role for CstF-50 in recognition of 3'-end processing signals.

Cleavage stimulation factor (CstF) is a highly conserved protein complex composed of three subunits that recognizes G/U-rich sequences downstream of the polyadenylation signal of eukaryotic mRNAs. While CstF has been identified over 25 years ago, the architecture and contribution of each subunit to RNA recognition have not been fully understood. In this study, we provide a structural basis for the recruitment of CstF-50 to CstF via interaction with CstF-77 and establish that the hexameric assembly of CstF creates a high affinity platform to target various G/U-rich sequences. We further demonstrate that CstF-77 boosts the affinity of the CstF-64 RRM to the RNA targets and CstF-50 fine tunes the ability of the complex to recognize G/U sequences of certain lengths and content.

Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection.

TDP-43 is a critical RNA-binding factor associated with pre-mRNA splicing in mammals. Its expression is tightly autoregulated, with loss of this regulation implicated in human neuropathology. We demonstrate that TDP-43 overexpression in humans and mice activates a 3' untranslated region (UTR) intron, resulting in excision of the proximal polyA site (PAS) pA(1). This activates a cryptic PAS that prevents TDP-43 expression through a nuclear retention mechanism. Superimposed on this process, overexpression of TDP-43 blocks recognition of pA(1) by competing with CstF-64 for PAS binding. Overall, we uncover complex interplay between transcription, splicing, and 3' end processing to effect autoregulation of TDP-43.

Hexameric architecture of CstF supported by CstF-50 homodimerization domain structure.

The Cleavage stimulation Factor (CstF) complex is composed of three subunits and is essential for pre-mRNA 3'-end processing. CstF recognizes U and G/U-rich cis-acting RNA sequence elements and helps stabilize the Cleavage and Polyadenylation Specificity Factor (CPSF) at the polyadenylation site as required for productive RNA cleavage. Here, we describe the crystal structure of the N-terminal domain of Drosophila CstF-50 subunit. It forms a compact homodimer that exposes two geometrically opposite, identical, and conserved surfaces that may serve as binding platform. Together with previous data on the structure of CstF-77, homodimerization of CstF-50 N-terminal domain supports the model in which the functional state of CstF is a heterohexamer.

The role of CSTF2 in cancer: from technology to clinical application.

A protein called cleavage-stimulating factor subunit 2 (CSTF2, additionally called CSTF-64) binds RNA and is needed for the cleavage and polyadenylation of mRNA. CSTF2 is an important component subunit of the cleavage stimulating factor (CSTF), which is located on the X chromosome and encodes 557 amino acids. There is compelling evidence linking elevated CSTF2 expression to the pathological advancement of cancer and on its impact on the clinical aspects of the disease. The progression of cancers, including hepatocellular carcinoma, melanoma, prostate cancer, breast cancer, and pancreatic cancer, is correlated with the upregulation of CSTF2 expression. This review provides a fresh perspective on the investigation of the associations between CSTF2 and various malignancies and highlights current studies on the regulation of CSTF2. In particular, the mechanism of action and potential clinical applications of CSTF2 in cancer suggest that CSTF2 can serve as a new biomarker and individualized treatment target for a variety of cancer types.

The tumor suppressor Cdc73 functionally associates with CPSF and CstF 3' mRNA processing factors.

The CDC73 tumor suppressor gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. Its product, the Cdc73 protein, is a component of the RNA polymerase II and chromatin-associated human Paf1 complex (Paf1C). Here, we show that Cdc73 physically associates with the cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF) complexes that are required for the maturation of mRNA 3' ends in the cell nucleus. Immunodepletion experiments indicate that the Cdc73-CPSF-CstF complex is necessary for 3' mRNA processing in vitro. Microarray analysis of CDC73 siRNA-treated cells revealed INTS6, a gene encoding a subunit of the Integrator complex, as an in vivo Cdc73 target. Cdc73 depletion by siRNA resulted in decreased INTS6 mRNA abundance, and decreased association of CPSF and CstF subunits with the INTS6 locus. Our results suggest that Cdc73 facilitates association of 3' mRNA processing factors with actively-transcribed chromatin and support the importance of links between tumor suppression and mRNA maturation.

Fip1 is a multivalent interaction scaffold for processing factors in human mRNA 3' end biogenesis.

3' end formation of most eukaryotic mRNAs is dependent on the assembly of a ~1.5 MDa multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3' end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal 'acidic' region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3' polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3' end processing.

The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation.

Because polyadenylation is essential for cell growth, in vivo examination of polyadenylation protein function has been difficult. Here we describe a new in vivo assay that allows structure-function assays on CstF-64, a protein that binds to pre-mRNAs downstream of the cleavage site for accurate and efficient polyadenylation. In this assay (the stem-loop luciferase assay for polyadenylation, SLAP), expression of a luciferase pre-mRNA with a modified downstream sequence element was made dependent upon co-expression of an MS2-CstF-64 fusion protein. We show here that SLAP accurately reflects CstF-64-dependent polyadenylation, confirming the validity of this assay. Using SLAP, we determined that CstF-64 domains involved in RNA binding, interaction with CstF-77 (the "Hinge" domain), and coupling to transcription are critical for polyadenylation. Further, we showed that the Hinge domain is necessary for CstF-64 interaction with CstF-77 and consequent nuclear localization, suggesting that nuclear import of a preformed CstF complex is an essential step in polyadenylation.

The structure of human cleavage factor I(m) hints at functions beyond UGUA-specific RNA binding: a role in alternative polyadenylation and a potential link to 5' capping and splicing.

3'-end cleavage and subsequent polyadenylation are critical steps in mRNA maturation. The precise location where cleavage occurs (referred to as poly(A) site) is determined by a tripartite mechanism in which a A(A/U)UAAA hexamer, GU rich downstream element and UGUA upstream element are recognized by the cleavage and polyadenylation factor (CPSF), cleavage stimulation factor (CstF) and cleavage factor I(m) (CFI(m)), respectively. CFI(m) is composed of a smaller 25 kDa subunit (CFI(m)25) and a larger 59, 68 or 72 kDa subunit. CFI(m)68 interacts with CFI(m)25 through its N-terminal RNA recognition motif (RRM). We recently solved the crystal structures of CFI(m)25 bound to RNA and of a complex of CFI(m)25, the RRM domain of CFI(m)68 and RNA. Our study illustrated the molecular basis for UGUA recognition by the CFI(m) complex, suggested a possible mechanism for CFI(m) mediated alternative polyadenylation, and revealed potential links between CFI(m) and other mRNA processing factors, such as the 20 kDa subunit of the cap binding protein (CBP20), and the splicing regulator U2AF65.

Pre-mRNA processing is partially impaired in satellite cell nuclei from aged muscles.

Satellite cells are responsible for the capacity of mature mammalian skeletal muscles to repair and maintain mass. During aging, skeletal muscle mass as well as the muscle strength and endurance progressively decrease, leading to a condition termed sarcopenia. The causes of sarcopenia are manifold and remain to be completely elucidated. One of them could be the remarkable decline in the efficiency of muscle regeneration; this has been associated with decreasing amounts of satellite cells, but also to alterations in their activation, proliferation, and/or differentiation. In this study, we investigated the satellite cell nuclei of biceps and quadriceps muscles from adult and old rats; morphometry and immunocytochemistry at light and electron microscopy have been combined to assess the organization of the nuclear RNP structural constituents involved in different steps of mRNA formation. We demonstrated that in satellite cells the RNA pathways undergo alterations during aging, possibly hampering their responsiveness to muscle damage.

The structure of the CstF-77 homodimer provides insights into CstF assembly.

The cleavage stimulation factor (CstF) is essential for the first step of poly(A) tail formation at the 3' ends of mRNAs. This heterotrimeric complex is built around the 77-kDa protein bridging both CstF-64 and CstF-50 subunits. We have solved the crystal structure of the 77-kDa protein from Encephalitozoon cuniculi at a resolution of 2 A. The structure folds around 11 Half-a-TPR repeats defining two domains. The crystal structure reveals a tight homodimer exposing phylogenetically conserved areas for interaction with protein partners. Mapping experiments identify the C-terminal region of Rna14p, the yeast counterpart of CstF-77, as the docking domain for Rna15p, the yeast CstF-64 homologue.

Structural insights into the 3'-end mRNA maturation machinery: Snapshot on polyadenylation signal recognition.

Pre-mRNA 3'-end maturation is achieved by a mechanism requiring four different protein complexes assembled from approximately twenty factors. A global understanding of this essential process is still missing due to the inability to structurally characterize the entire complexes, even though structures of the isolated factors have been obtained. In this review, we summarize recent findings regarding the atomic description of one of the major players, the Cleavage and Polyadenylation Specificity Factor complex (CPSF in human, CPF in yeast). These data provide information on the architecture adopted by the major components of this complex, and on its capacity to recognize the polyadenylation signal sequence.

The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50.

The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

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.

Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers.

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, tauCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between tauCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of tauCstF-64 and CstF-64 have different affinities for RNA elements. We quantified K(d) values of CstF-64 and tauCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than tauCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal alpha-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is mportant in RNA sequence recognition. This supports the hypothesis that tauCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements.

The use of in situ proteolysis in the crystallization of murine CstF-77.

The cleavage-stimulation factor (CstF) is required for the cleavage of the 3'-end of messenger RNA precursors in eukaryotes. During structure determination of the 77 kDa subunit of the murine CstF complex (CstF-77), it was serendipitously discovered that a solution infected by a fungus was crucial for the crystallization of this protein. CstF-77 was partially proteolyzed during crystallization; this was very likely to have been catalyzed by a protease secreted by the fungus. It was found that the fungal protease can be replaced by subtilisin and this in situ proteolysis protocol produced crystals of sufficient size for structural studies. After an extensive search, it was found that 55% glucose can be used as a cryoprotectant while maintaining the diffraction quality of the crystals; most other commonly used cryoprotectants were detrimental to the diffraction quality.

Delineating the structural blueprint of the pre-mRNA 3'-end processing machinery.

Processing of mRNA precursors (pre-mRNAs) by polyadenylation is an essential step in gene expression. Polyadenylation consists of two steps, cleavage and poly(A) synthesis, and requires multiple cis elements in the pre-mRNA and a megadalton protein complex bearing the two essential enzymatic activities. While genetic and biochemical studies remain the major approaches in characterizing these factors, structural biology has emerged during the past decade to help understand the molecular assembly and mechanistic details of the process. With structural information about more proteins and higher-order complexes becoming available, we are coming closer to obtaining a structural blueprint of the polyadenylation machinery that explains both how this complex functions and how it is regulated and connected to other cellular processes.