Cloning and characterization of Arabidopsis homologues of the animal CstF complex that regulates 3' mRNA cleavage and polyadenylation

The 3' cleavage and polyadenylation of mRNAs has been studied in detail in animals and yeast, but not in plants. Aimed at elucidating the regulation of mRNA 3' end formation in plants, three Arabidopsis cDNAs encoding homologues of the animal proteins CstF-64, CstF-77 and CstF-50 that form the cleavage stimulating factor of the polyadenylation machinery have been cloned. It is shown experimentally that the N-terminal domain of the Arabidopsis CstF-64 homologue binds the mRNA 3' non-coding region in an analogous manner to the animal protein. It is also shown that the Arabidopsis CstF-64 and CstF-77 homologues strongly interact with each other in a similar way to their animal counterparts. These results imply that these Arabidopsis homologues belong to the polyadenylation machinery of nuclear mRNAs.     

An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing.     

Locked tether formation by cooperative folding of Rna14p monkeytail and Rna15p hinge domains in the yeast CF IA complex

The removal of the 3' region of pre-mRNA followed by polyadenylation is a key step in mRNA maturation. In the yeast Saccharomyces cerevisiae, one component of the processing machinery is the cleavage/polyadenylation factor IA (CF IA) complex, composed of four proteins (Clp1p, Pcf11p, Rna14p, Rna15p) that recognize RNA sequences adjacent to the cleavage site and recruit additional processing factors. To gain insight into the molecular architecture of CF IA we solved the solution structure of the heterodimer composed of the interacting regions between Rna14p and Rna15p. The C-terminal monkeytail domain from Rna14p and the hinge region from Rna15p display a coupled binding and folding mechanism, where both peptides are initially disordered. Mutants with destabilized monkeytail-hinge interactions prevent association of Rna15p within CF IA. Conservation of interdomain residues reveals that the structural tethering is preserved in the homologous mammalian cleavage stimulation factor (CstF)-77 and CstF-64 proteins of the CstF complex.     

Formation of the 3' end of histone mRNA: getting closer to the end

Nearly all eukaryotic mRNAs end with a poly(A) tail that is added to their 3' end by the ubiquitous cleavage/polyadenylation machinery. The only known exceptions to this rule are metazoan replication-dependent histone mRNAs, which end with a highly conserved stem-loop structure. This distinct 3' end is generated by specialized 3' end processing machinery that cleaves histone pre-mRNAs 4-5 nucleotides downstream of the stem-loop and consists of the U7 small nuclear RNP (snRNP) and number of protein factors. Recently, the U7 snRNP has been shown to contain a unique Sm core that differs from that of the spliceosomal snRNPs, and an essential heat labile processing factor has been identified as symplekin. In addition, cross-linking studies have pinpointed CPSF-73 as the endonuclease, which catalyzes the cleavage reaction. Thus, many of the critical components of the 3' end processing machinery are now identified. Strikingly, this machinery is not as unique as initially thought but contains at least two factors involved in cleavage/polyadenylation, suggesting that the two mechanisms have a common evolutionary origin. The greatest challenge that lies ahead is to determine how all these factors interact with each other to form a catalytically competent processing complex capable of cleaving histone pre-mRNAs.     

A CPSF-73 homologue is required for cell cycle progression but not cell growth and interacts with a protein having features of CPSF-100

Formation of the mature 3' ends of the vast majority of cellular mRNAs occurs through cleavage and polyadenylation and requires a cleavage and polyadenylation specificity factor (CPSF) containing, among other proteins, CPSF-73 and CPSF-100. These two proteins belong to a superfamily of zinc-dependent beta-lactamase fold proteins with catalytic specificity for a wide range of substrates including nucleic acids. CPSF-73 contains a zinc-binding histidine motif involved in catalysis in other members of the beta-lactamase superfamily, whereas CPSF-100 has substitutions within the histidine motif and thus is unlikely to be catalytically active. Here we describe two previously unknown human proteins, designated RC-68 and RC-74, which are related to CPSF-73 and CPSF-100 and which form a complex in HeLa and mouse cells. RC-68 contains the intact histidine motif, and hence it might be a functional counterpart of CPSF-73, whereas RC-74 lacks this motif, thus resembling CPSF-100. In HeLa cells RC-68 is present in both the cytoplasm and the nucleus whereas RC-74 is exclusively nuclear. RC-74 does not interact with CPSF-73, and neither RC-68 nor RC-74 is found in a complex with CPSF-160, indicating that these two proteins form a separate entity independent of the CPSF complex and are likely involved in a pre-mRNA processing event other than cleavage and polyadenylation of the vast majority of cellular pre-mRNAs. RNA interference-mediated depletion of RC-68 arrests HeLa cells early in G(1) phase, but surprisingly the arrested cells continue growing and reach the size typical of G(2) cells. RC-68 is highly conserved from plants to humans and may function in conjunction with RC-74 in the 3' end processing of a distinct subset of cellular pre-mRNAs encoding proteins required for G(1) progression and entry into S phase.     

Polyadenylation site-specific differences in the activity of the neuronal βCstF-64 protein in PC-12 cells

Recent genome-wide analyses have implicated alternative polyadenylation — the process of regulated mRNA 3′ end formation — as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, βCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that βCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for βCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that βCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that βCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the β-adducin mRNA. Notably, we demonstrate that the activity of βCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the βCstF-64 protein. Our data address the polyadenylation functions of βCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.     

CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3′ end processing

Embryonic stem cells (ESCs) exhibit a unique cell cycle with a shortened G₁ phase that supports their pluripotency, while apparently buffering them against pro-differentiation stimuli. In ESCs, expression of replication-dependent histones is a main component of this abbreviated G₁ phase, although the details of this mechanism are not well understood. Similarly, the role of 3′ end processing in regulation of ESC pluripotency and cell cycle is poorly understood. To better understand these processes, we examined mouse ESCs that lack the 3′ end-processing factor CstF-64. These ESCs display slower growth, loss of pluripotency and a lengthened G₁ phase, correlating with increased polyadenylation of histone mRNAs. Interestingly, these ESCs also express the τCstF-64 paralog of CstF-64. However, τCstF-64 only partially compensates for lost CstF-64 function, despite being recruited to the histone mRNA 3′ end-processing complex. Reduction of τCstF-64 in CstF-64-deficient ESCs results in even greater levels of histone mRNA polyadenylation, suggesting that both CstF-64 and τCstF-64 function to inhibit polyadenylation of histone mRNAs. These results suggest that CstF-64 plays a key role in modulating the cell cycle in ESCs while simultaneously controlling histone mRNA 3′ end processing.     

Early evolution of histone mRNA 3' end processing

The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.     

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.