Crystal structure of a human cleavage factor CFI(m)25/CFI(m)68/RNA complex provides an insight into poly(A) site recognition and RNA looping

Cleavage factor I(m) (CFI(m)) is a highly conserved component of the eukaryotic mRNA 3' processing machinery that functions in sequence-specific poly(A) site recognition through the collaboration of a 25 kDa subunit containing a Nudix domain and a larger subunit of 59, 68, or 72 kDa containing an RNA recognition motif (RRM). Our previous work demonstrated that CFI(m)25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here, we report the crystal structure of CFI(m)25 complexed with the RRM domain of CFI(m)68 and RNA. The CFI(m)25 dimer is clasped on opposite sides by two CFI(m)68 RRM domains. Each CFI(m)25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFI(m)68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFI(m) to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.     

Mpe1, a Zinc Knuckle Protein, Is an Essential Component of Yeast Cleavage and Polyadenylation Factor Required for the Cleavage and Polyadenylation of mRNA

In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene, MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice of ACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3'-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3' end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3' end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.     

Getting in the loop: new insights into the mechanism of poly(A) site recognition

In this issue, Yang et al. (2011) show that the 3' end processing factor CFI(m) interacts with RNA in manner that facilitates RNA looping, suggesting mechanistic roles for this factor in the regulation of poly(A) site selection.     

RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor.

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.     

Specific pre-cleavage and post-cleavage complexes involved in the formation of SV40 late mRNA 3'' termini in vitro.

Complexes form between processing factors present in a crude nuclear extract from HeLa cells and a simian virus 40 (SV40) late pre-mRNA which spans the polyadenylation [poly(A)] site. A specific 'pre-cleavage complex' forms on the pre-mRNA before cleavage. Formation of this complex requires the highly conserved sequence AAUAAA: it is prevented by mutations in AAUAAA, and by annealing DNA oligonucleotides to that sequence. After cleavage, the 5' half-molecule is found in a distinct 'post-cleavage complex'. In contrast, the 3' half-molecule is released. After cleavage and polyadenylation, polyadenylated RNA also is released. De novo formation of the post-cleavage complex requires AAUAAA and a nearby 3' terminus. Competition experiments suggest that a component which recognizes AAUAAA is required for formation of both pre- and post-cleavage complexes.     

Interaction of poly(A) polymerase with the 25-kDa subunit of cleavage factor I.

Mammalian poly(A) polymerase (PAP), a key enzyme in the pre-mRNA 3'-end processing reaction, carries the catalytic domain in the N-terminal region, an RNA binding domain, two nuclear localization signals, and a serine/threonine-rich regulatory domain in the C-terminal region. Using LexA-based yeast two-hybrid screening, we identified a cDNA encoding the 25-kDa subunit of cleavage factor I (CFI-25) as a protein that interacts with the C-terminal region of mouse PAP. The glutathione S-transferase pull-down assay and the immunoprecipitation experiment revealed that PAP directly interacts with CFI-25 and that the C-terminal 69 residues of PAP and the N-terminal 60 residues of CFI-25 are sufficient for the interaction between CFI-25 and PAP. Since CFI is known to function in the assembly of the pre-mRNA 3'-processing complex, this interaction may play an important role in the assembly of the processing complex and/or in the regulation of PAP activity within the complex.     

Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus.

Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA.     

Conventional 3' end formation is not required for NMD substrate recognition in Saccharomyces cerevisiae

The recognition and rapid degradation of mRNAs with premature translation termination codons by the nonsense-mediated pathway of mRNA decay is an important RNA quality control system in eukaryotes. In mammals, the efficient recognition of these mRNAs is dependent upon exon junction complex proteins deposited on the RNA during pre-mRNA splicing. In yeast, splicing does not play a role in recognition of mRNAs that terminate translation prematurely, raising the possibility that proteins deposited during alternative pre-mRNA processing events such as 3' end formation might contribute to the distinction between normal and premature translation termination. We have utilized mRNAs with a 3' poly(A) tail generated by ribozyme cleavage to demonstrate that the normal process of 3' end cleavage and polyadenylation is not required for mRNA stability or the detection of a premature stop codon. Thus, in yeast, the distinction between normal and premature translation termination events is independent of both splicing and conventional 3' end formation.     

Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing

Biochemical components (splicing factors) interact with specific intron regions during pre-mRNA splicing in vitro. The pre-mRNA specifically associates with factors at both the branch point and the 5' splice site and these RNA-factor interactions are maintained in the intron-containing RNA processing products. The first detectable event, the ATP-dependent association of a factor (or factors) with the branch point, is mediated by at least one factor containing an essential nucleic acid component. Mutant RNA substrates that lack either the 5' splice site or the vast majority of exon sequences can still associate with the branch point binding factor(s). However, this branch point-factor interaction does not occur with a mutant RNA substrate that contains the branch point but that lacks the 3' splice site consensus sequence. These results suggest that selection of the 3' splice site accompanied by the association of a factor with the branch point may be the initial step in mammalian pre-mRNA splicing.     

Formation of mRNA 3'' termini: stability and dissociation of a complex involving the AAUAAA sequence.

Formation of the 3' termini of mRNAs in animal cells involves endonucleolytic cleavage of a pre-mRNA, followed by polyadenylation of the newly formed end. Here we demonstrate that, during cleavage in vitro, the highly conserved AAUAAA sequence of the pre-mRNA forms a complex with a factor present in a crude nuclear extract. This complex is required for cleavage and polyadenylation. It normally is transient, but is very stable on cleaved RNA to which a single terminal cordycepin residue has been added. The complex can form either during the cleavage reaction, or on a synthetic RNA that ends at the polyadenylation site. Mutations which prevent cleavage also prevent complex formation. The complex dissociates during or after polyadenylation, enabling the released activities to catalyze a second round of cleavage.     

A novel plant in vitro assay system for pre-mRNA cleavage during 3'-end formation

Messenger RNA (mRNA) maturation in eukaryotic cells requires the formation of the 3' end, which includes two tightly coupled steps: the committing cleavage reaction that requires both correct cis-element signals and cleavage complex formation, and the polyadenylation step that adds a polyadenosine [poly(A)] tract to the newly generated 3' end. An in vitro biochemical assay plays a critical role in studying this process. The lack of such an assay system in plants hampered the study of plant mRNA 3'-end formation for the last two decades. To address this, we have now established and characterized a plant in vitro cleavage assay system, in which nuclear protein extracts from Arabidopsis (Arabidopsis thaliana) suspension cell cultures can accurately cleave different pre-mRNAs at expected in vivo authenticated poly(A) sites. The specific activity is dependent on appropriate cis-elements on the substrate RNA. When complemented by yeast (Saccharomyces cerevisiae) poly(A) polymerase, about 150-nucleotide poly(A) tracts were added specifically to the newly cleaved 3' ends in a cooperative manner. The reconstituted polyadenylation reaction is indicative that authentic cleavage products were generated. Our results not only provide a novel plant pre-mRNA cleavage assay system, but also suggest a cross-kingdom functional complementation of yeast poly(A) polymerase in a plant system.