A mechanism for the regulation of pre-mRNA 3' processing by human cleavage factor Im

Human cleavage factor I(m) (CFI(m)) is a heterodimeric RNA binding protein complex that functions at an early step in the assembly of the pre-mRNA 3' processing complex. In this report we show that CFI(m) can stimulate both cleavage and poly(A) addition, and can act to suppress poly(A) site cleavage in a sequence-dependent manner. Elevated levels of CFI(m) suppressed cleavage at the primary poly(A) site of the pre-mRNA encoding the 68 kDa subunit of CFI(m). CFI(m)-mediated suppression of poly(A) site cleavage was dependent upon the presence of three copies of an RNA element initially identified by CFI(m)-SELEX. These data provide evidence for a mechanism for the regulation of poly(A) site selection by a basal pre-mRNA 3' processing factor.     

Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation

tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.     

Analysis and recognition of 5' UTR intron splice sites in human pre-mRNA

Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites and other features found in the adjacent exons and introns. By restricting the training of neural network algorithms to 'pure' UTRs (not extending partially into protein coding regions), we for the first time investigate the predictive power of the splicing signal proper, in contrast to conventional splice site prediction, which typically relies on the change in sequence at the transition from protein coding to non-coding. By doing so, the algorithms were able to pick up subtler splicing signals that were otherwise masked by 'coding' noise, thus enhancing significantly the prediction of 5' UTR splice sites. For example, the non-coding splice site predicting networks pick up compositional and positional bias in the 3' ends of non-coding exons and 5' non-coding intron ends, where cytosine and guanine are over-represented. This compositional bias at the true UTR donor sites is also visible in the synaptic weights of the neural networks trained to identify UTR donor sites. Conventional splice site prediction methods perform poorly in UTRs because the reading frame pattern is absent. The NetUTR method presented here performs 2-3-fold better compared with NetGene2 and GenScan in 5' UTRs. We also tested the 5' UTR trained method on protein coding regions, and discovered, surprisingly, that it works quite well (although it cannot compete with NetGene2). This indicates that the local splicing pattern in UTRs and coding regions is largely the same. The NetUTR method is made publicly available at www.cbs.dtu.dk/services/NetUTR.     

Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

Mammalian cleavage factor I (CF I_m) is composed of two polypeptides of 25 kDa and either a 59 or 68 kDa subunit (CF I_m25, CF I_m59, CF I_m68). It is part of the cleavage and polyadenylation complex responsible for processing the 3′ ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 3′ processing complex, we systematically searched for enzymes that modify arginines by the addition of methyl groups. Protein arginine methyltransferases (PRMTs) are such enzymes that transfer methyl groups from S-adenosyl methionine to arginine residues within polypeptide chains resulting in mono- or dimethylated arginines. We found that CF I_m68 and the nuclear poly(A) binding protein 1 (PABPN1) were methylated by HeLa cell extracts in vitro. By fractionation of these extracts followed by mass spectral analysis, we could demonstrate that the catalytic subunit PRMT5, together with its cofactor WD45, could symmetrically dimethylate CF I_m68, whereas pICln, the third polypeptide of the complex, was stimulatory. As sites of methylation in CF I_m68 we could exclusively identify arginines in a GGRGRGRF or “GAR” motif that is conserved in vertebrates. Further in vitro assays revealed a second methyltransferase, PRMT1, which modifies CF I_m68 by asymmetric dimethylation of the GAR motif and also weakly methylates the C-termini of both CF I_m59 and CF I_m68. The results suggest that native—as compared with recombinant—protein substrates may contain additional determinants for methylation by specific PRMTs. A possible involvement of CF I_m methylation in the context of RNA export is discussed.     

Variable effects of the conserved RNA hairpin element upon 3' end processing of histone pre-mRNA in vitro.

We have studied the requirements for efficient histone-specific RNA 3' processing in nuclear extract from mammalian tissue culture cells. Processing is strongly impaired by mutations in the pre-mRNA spacer element that reduce the base-pairing potential with U7 RNA. Moreover, by exchanging the hairpin and spacer elements of two differently processed H4 genes, we find that this difference is exclusively due to the spacer element. Finally, processing is inhibited by the addition of competitor RNAs, if these contain a wild-type spacer sequence, but not if their spacer element is mutated. Conversely, the importance of the hairpin for histone RNA 3' processing is highly variable: A hairpin mutant of the H4-12 gene is processed with almost wild-type efficiency in extract from K21 mouse mastocytoma cells but is strongly affected in HeLa cell extract, whereas an identical hairpin mutant of the H4-1 gene is affected in both extracts. The hairpin defect of H4-12-specific RNA in HeLa cells can be overcome by a compensatory mutation that increases the base complementarity to U7 snRNA. Very similar results were also obtained in RNA competition experiments: processing of H4-12-specific RNA can be competed by RNA carrying a wild-type hairpin element in extract from HeLa, but not K21 cells, whereas processing of H4-1-specific RNA can be competed in both extracts. With two additional histone genes we obtained results that were in one case intermediate and in the other similar to those obtained with H4-1. These results suggest that hairpin binding factor(s) can cooperatively support the ability of U7 snRNPs to form an active processing complex, but is(are) not directly involved in the processing mechanism.     

Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA.

Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.     

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

The cleavage factor I(m) (CF I(m)), consists of a 25 kDa subunit (CF I(m)25) and one of three larger subunits (CF I(m)59, CF I(m)68, CF I(m)72), and is an essential protein complex for pre-mRNA 3'-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF I(m), comprising CF I(m)25 and the RNA recognition motif domain of CF I(m)68 (CF I(m)68RRM), and the crystal structure of the CF I(m)-RNA complex. These structures show that two CF I(m)68RRM molecules bind to the CF I(m)25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF I(m)25-CF I(m)68RRM heterotetramer, providing structural basis for the mechanism by which CF I(m) binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF I(m)68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF I(m)68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF I(m)68 makes an essential contribution to pre-mRNA binding.     

Requirements of the RNA polymerase II C-terminal domain for reconstituting pre-mRNA 3' cleavage

RNA polymerase II (RNAP II) has previously been shown to be required for the pre-mRNA polyadenylation cleavage reaction in vitro. This activity was found to reside solely in the C-terminal domain (CTD) of the enzyme's largest subunit. Using a deletion analysis of glutathione S-transferase-CTD fusion proteins, we searched among the CTD's 52 imperfectly repetitive heptapeptides for the minimal subset that possesses this property. We found that heptads in the vicinity of 30 to 37 contribute modestly more than other sections, but that no specific subsection of the CTD is necessary or sufficient for cleavage. To investigate further the heptad requirements for cleavage, we constructed a series of all-consensus CTDs having 13, 26, 39, and 52 YSPTSPS repeats. We found that the nonconsensus CTD heptads are together responsible for only 20% of the wild-type cleavage activity. Analysis of the all-consensus CTD series revealed that the remaining 80% of the CTD-dependent cleavage activity directly correlates with CTD length, with significant activity requiring approximately 26 or more repeats. These results are consistent with a scaffolding role for the RNAP II CTD in the pre-mRNA cleavage reaction.     

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.