Participation of the nuclear cap binding complex in pre-mRNA 3' processing.

Communication between the 5' and 3' ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5' end is bound by the nuclear cap binding complex (CBC). This RNA-protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3' end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5' and 3' ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3' processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of "closed-loops," due to interactions between factors bound at their 5' and 3' ends.

Restoration of both structure and function to a defective poly(A) site by in vitro selection.

Efficient cleavage and polyadenylation at the human immunodeficiency virus type-1 (HIV-1) poly(A) site requires an upstream 3'-processing enhancer to overcome the suboptimal sequence context of the AAUAAA hexamer. The HIV-1 3'-processing enhancer functions to stabilize the association of the pre-mRNA with cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of the AAUAAA hexamer. Intriguingly, in the absence of the 3'-processing enhancer, CPSF binding and polyadenylation efficiency could be restored to near wild-type levels upon replacement of the 14-nucleotide region immediately 5' of the HIV-1 AAUAAA hexamer (the B segment) by the analogous sequences from the efficient adenovirus L3 poly(A) site. To further investigate the contributions of RNA sequence and structure to poly(A) site recognition, we have used an in vitro selection system to identify B segment sequences that enhance the polyadenylation efficiency of a pre-cleaved RNA lacking a 3'-processing enhancer. The final RNA selection pool was composed of two predominant classes of RNAs. Nuclease probing revealed that the selected sequences restored an RNA conformation that facilitates recognition of the AAUAAA hexamer by CPSF. These results indicate that both the sequence and structural context of the AAUAAA hexamer contribute to poly(A) site recognition by CPSF.

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.

A common mechanism for the enhancement of mRNA 3' processing by U3 sequences in two distantly related lentiviruses.

The protein coding regions of all retroviral pre-mRNAs are flanked by a direct repeat of R-U5 sequences. In many retroviruses, the R-U5 repeat contains a complete core poly(A) site-composed of a highly conserved AAUAAA hexamer and a GU-rich downstream element. A mechanism that allows for the bypass of the 5' core poly(A) site and the exclusive use of the 3' core poly(A) site must therefore exist. In human immunodeficiency virus type 1 (HIV-1), sequences within the U3 region appear to play a key role in poly(A) site selection. U3 sequences are required for efficient 3' processing at the HIV-1 poly(A) site both in vivo and in vitro. These sequences serve to promote the interaction of cleavage and polyadenylation specificity factor (CPSF) with the core poly(A) site. We have now demonstrated the presence of a functionally analogous 3' processing enhancer within the U3 region of a distantly related lentivirus, equine infectious anemia virus (EIAV). U3 sequences enhanced the processing of the EIAV core poly(A) site sevenfold in vitro. The U3 sequences also enhanced the stability of CPSF binding at the core poly(A) site. Optimal processing required the TAR RNA secondary structure that resides within the R region 28 nucleotides upstream of the AAUAAA hexamer. Disruption of TAR reduced processing, while compensatory changes that restored the RNA structure also restored processing to the wild-type level, suggesting a position dependence of the U3-encoded enhancer sequences. Finally, the reciprocal exchange of the EIAV and HIV U3 regions demonstrated the ability of each of these sequences to enhance both 3' processing and the binding of CPSF in the context of the heterologous core poly(A) site. The impact of U3 sequences upon the interaction of CPSF at the core poly(A) site may therefore represent a common strategy for retroviral poly(A) site selection.

Sequences regulating poly(A) site selection within the adenovirus major late transcription unit influence the interaction of constitutive processing factors with the pre-mRNA.

The adenovirus major late transcription unit (MLTU) encodes five families of mRNAs, L1 to L5, each distinguished by a unique poly(A) site. Use of the promoter-proximal L1 poly(A) site predominates during early infection, whereas poly(A) site choice shifts to the promoter-distal sites during late infection. A mini-MLTU containing only the L1 and L3 poly(A) sites has been shown to reproduce this processing switch. In vivo analysis has revealed that sequences extending 5' and 3' of the L1 core poly(A) site are required for efficient processing as well as for regulated expression. By replacement of the L1 core poly(A) site with that of the ground squirrel hepatitis virus poly(A) site, we now demonstrate that the L1 flanking sequences can enhance the processing of a heterologous poly(A). Upon recombination of the chimeric L1-ground squirrel hepatitis virus poly(A) site onto the viral chromosome, the L1 flanking sequences were also found to be sufficient to reproduce the processing switch during the course of viral infection. Subsequent in vitro analysis has shown that the L1 flanking sequences function to enhance the stability of binding of cleavage and polyadenylation specificity factor to the core poly(A) site. The impact of L1 flanking sequences on the binding of cleavage and polyadenylation specificity factor suggests that the regulation of the MLTU poly(A) site selection is mediated by the interaction of constitutive processing factors.

CPSF recognition of an HIV-1 mRNA 3'-processing enhancer: multiple sequence contacts involved in poly(A) site definition.

The endonucleolytic cleavage and polyadenylation of a pre-mRNA in mammalian cells requires two cis-acting elements, a highly conserved AAUAAA hexamer and an amorphous U- or GU-rich downstream element, that together constitute the "core" poly(A) site. The terminal redundancy of the HIV-1 pre-mRNA requires that the processing machinery disregard a core poly(A) site at the 5' end of the transcript, and efficiently utilize an identical signal that resides near the 3' end. Efficient processing at the 3' core poly(A) site, both in vivo and in vitro, has been shown to require sequences 76 nucleotides upstream of the AAUAAA hexamer. In this report we demonstrate that this HIV-1 upstream element interacts directly with the 160-kD subunit of CPSF (cleavage polyadenylation specificity factor), the factor responsible for the recognition of the AAUAAA hexamer. The presence of the upstream element in the context of the AAUAAA hexamer directs the stable binding of CPSF to the pre-mRNA and enhances the efficiency of poly(A) addition in reactions reconstituted with purified CPSF and recombinant poly(A) polymerase. Our results indicate that the dependence of HIV-1 3' processing on upstream sequences is a consequence of the suboptimal sequence context of the AAUAAA hexamer. We suggest that poly(A) site definition involves the recognition of multiple heterogeneous sequence elements in the context of the AAUAAA hexamer.

Activation of HIV-1 pre-mRNA 3' processing in vitro requires both an upstream element and TAR.

The architecture of the human immunodeficiency virus type 1 (HIV-1) genome presents an intriguing dilemma for the 3' processing of viral transcripts--to disregard a canonical 'core' poly(A) site processing signal present at the 5' end of the transcript and yet to utilize efficiently an identical signal that resides at the 3' end of the message. The choice of processing sites in HIV-1 appears to be influenced by two factors: (i) proximity to the cap site, and (ii) sequences upstream of the core poly(A) site. We now demonstrate that an in vivo-defined upstream element that resides within the U3 region, 76 nucleotides upstream of the AAUAAA hexamer, acts specifically to enhance 3' processing at the HIV-1 core poly(A) site in vitro. We furthermore show that efficient in vitro 3' processing requires the RNA stem-loop structure of TAR, which serves to juxtapose spatially the upstream element and the core poly(A) site. An analysis of the stability of 3' processing complexes formed at the HIV-1 poly(A) site in vitro suggests that the upstream element may function by increasing processing complex stability at the core poly(A) site.

Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA.

Poly(A) site processing of a pre-mRNA requires the participation of multiple nuclear factors. Two of these factors recognize specific sequences in the pre-mRNA and form a stable processing complex. Since these initial interactions are likely critical for the recognition of the poly(A) site and the efficiency of poly(A) site use, we have characterized these factors and the nature of their interaction with the pre-mRNA. The AAUAAA specificity factor PF2 is a large, multicomponent complex composed of at least five distinct polypeptides ranging in molecular size from 170 to 42 kDa. The 170-kDa polypeptide appears to mediate interaction with the pre-mRNA. Factor CF1, which provides specificity for the downstream G + U-rich element and stabilizes the PF2 interaction on the RNA, is also a multicomponent complex but is less complex than PF2. CF1 is composed of three polypeptides of molecular sizes 76, 64, and 48 kDa. UV cross-linking assays demonstrate that the 64-kDa polypeptide makes direct contact with the RNA, dependent on the G + U-rich downstream sequence element. Moreover, it is clear that these RNA-protein interactions are influenced by the apparent cooperative interaction involving PF2 and CF1, interactions that contribute to the efficiency of poly(A) site processing.

The HTLV-I Rex response element mediates a novel form of mRNA polyadenylation.

HTLV-I structural gene expression is posttranscriptionally regulated by the Rex protein and the Rex response element (RexRE), a 255 nucleotide RNA stem-loop structure located in the retroviral 3' LTR. Independent of Rex, the RexRE also plays a critical role in the polyadenylation of all HTLV-I transcripts. Folding of the RexRE serves to spatially juxtapose the widely separated AAUAAA hexamer and GU-rich elements that are essential for polyadenylation. In turn, this folding promotes the cooperative and stable binding of two nuclear factors at these elements that commits this poly(A) site to 3' processing. These findings highlight a novel mechanism of 3' end formation in the HTLV family of retroviruses and underscore the general requirement for protein-protein interactions in the polyadenylation reaction.

Poly(A) site efficiency reflects the stability of complex formation involving the downstream element.

A critical step in mRNA biogenesis is the generation of the mRNA 3' end through an endonucleolytic cleavage of the primary transcript followed by the addition of a approximately 200 nucleotide (nt) poly(A) tail. The efficiency of poly(A) site function can vary widely and for those genes with multiple poly(A) sites, the choice can be a regulated event. A functional poly(A) site is characterized by cis-acting RNA sequences including the well-conserved AAUAAA hexamer, located 10-30 nt upstream of the cleavage site, and a highly variable downstream GU- or U-rich element. The gene specific nature of the downstream sequence suggests that it may be a primary determinant of poly(A) site efficiency. Several recent studies have detailed the purification of factors that mediate the cleavage and polyadenylation reaction and that recognize the cis-acting signals. Two of these factors are responsible for the formation of a stable, committed ternary complex with the pre-RNA. In order to define the role of this stable complex in poly(A) site function, we have compared the processing efficiency of several pre-mRNAs with the stability of the complex that forms on these RNAs. We show that ternary complex stability reflects both the in vivo and the in vitro efficiency of the poly(A) site and that the stability of this complex is dependent on the nature of the downstream sequence element. We conclude that the stability of these protein--RNA interactions, dictated by the downstream element, plays a major role in determining the processing efficiency of a particular poly(A) site.

An ordered pathway of assembly of components required for polyadenylation site recognition and processing.

Four HeLa cell nuclear factors that are required for specific pre-mRNA cleavage and polyadenylation have been extensively purified, thereby permitting an investigation of the role of each in the 3' processing reaction. Two factors, termed PF1 and PF2, are required for specific polyadenylation of the cleaved RNA. PF1 is a poly(A) polymerase, and PF2 is a factor that confers AAUAAA specificity to the reaction. Both of these factors, along with two additional factors termed CF1 and CF2, are required for the endonucleolytic cleavage of the pre-mRNA. The ability of each of these factors to form specific complexes with the pre-mRNA was assayed using native gel electrophoresis. Two distinct complexes were detected. PF2 forms an initial complex with the pre-RNA, dependent on the AAUAAA sequence element but independent of specific downstream sequences. Formation of the PF2-RNA complex permits the subsequent interaction of CF1 and the formation of a second, larger complex. CF1 binding requires the downstream sequence element in addition to PF2 binding. Whereas the PF2-RNA complex is unstable and dissociates rapidly, the ternary complex formed by CF1, PF2, and RNA is stable. Thus, the interaction of CF1, dependent on the downstream sequence element, can be viewed as a commitment of the poly(A) site for processing. On the addition of the poly(A) polymerase (PF1) and factor CF2, the pre-mRNA is specifically cleaved at the poly(A) site.

Multiple factors are required for poly(A) addition to a mRNA 3' end.

Polyadenylation of pre-mRNAs in the nucleus involves a specific endonucleolytic cleavage, followed by the addition of approximately 200 adenylic acid residues. We have assayed HeLa nuclear extracts for the activity that catalyzes the poly(A) addition reaction. The authenticity of the in vitro assay was indicated by the observation that the poly(A) tract added in vitro is approximately 200 nucleotides in length. We have fractionated nuclear extracts in order to define components involved in specific poly(A) addition. No single fraction from DEAE-Sephacel chromatography of a HeLa nuclear extract possessed the specific poly(A) addition activity. However, if the various fractions were recombined, activity was restored, indicating the presence of multiple components. Further fractionation revealed the presence of at least two factors necessary for the poly(A) addition reaction. The reconstituted system retains the characteristics and specificity seen in the crude extract. Additional purification of one of the factors strongly suggests it to be a previously characterized poly(A) polymerase which, when assayed in the absence of the other factor, can add AMP to an RNA terminus but without specificity. Thus, the other component of the reaction may provide specificity to the process. In contrast to the 3' cleavage reaction, the poly(A) addition machinery does not possess an essential RNA component, as assayed by micrococcal nuclease digestion, nor do anti-Sm sera inhibit the reaction. Thus, the total process of formation of a polyadenylated mRNA 3' end is complex and requires the concerted action of distinct nuclear components.

Multiple factors are required for specific RNA cleavage at a poly(A) addition site.

An SP6 RNA containing the adenovirus 5 L3 poly(A) site is processed efficiently in a HeLa cell nuclear extract to generate correct 3' termini. Accurate 3' processing has also been demonstrated for the adenovirus E2A and SV40 early poly(A) sites, although these are processed less efficiently than the L3 site. Efficient cleavage at the poly(A) site requires the presence of a 5'-cap structure, as well as the RNA sequence motifs previously shown to be necessary for 3' processing in vivo, suggesting the presence and action of the appropriate factors in the nuclear extract. Fractionation of the nuclear extract has revealed a requirement for at least two distinct factors for cleavage at the L3 poly(A) site. One of these factors appears to possess an RNA component due to its sensitivity to micrococcal nuclease. The activity of this fraction is also sensitive to alpha-Sm monoclonal antibody, indicating the presence of an snRNP essential for the cleavage reaction. Additional factors are required for the subsequent polyadenylation reaction, indicating the involvement of a multicomponent complex in the processing of an RNA at the poly(A) site.

Functional analysis of the sea urchin U7 small nuclear RNA.

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 3' end of H3 messenger RNA.

Processing of the 3' end of sea urchin H3 histone pre-mRNA requires conserved sequence elements and the presence of U7 snRNA. A mutation in the conserved CAAGAAGA sequence of the H3 pre-mRNA that renders 3' processing of this precursor defective is shown to be suppressed by a compensatory change in the U7 snRNA, restoring the base-pairing potential of the two RNAs. RNA-RNA contacts between these two molecules appear to be an essential feature of the 3' processing reaction.

Identification of transcriptional elements within the long terminal repeat of Rous sarcoma virus.

Transcriptional regulatory elements within the Rous sarcoma virus long terminal repeat were examined by the construction of a series of deletions and small insertions within the U3 region of the long terminal repeat. The analysis of these mutations in chicken embryo cells and COS cells permitted the identification of important transcriptional regulatory elements. Sequences within the region 31 to 18 base pairs upstream of the RNA cap site (-31 to -18), encompassing a TATA box-like sequence, function in the selection of the correct site of transcription initiation and, in addition, augment the efficiency of transcription. These sequences are essential for virus replication. Sequences within the region -79 to -59, overlapping a CAAT box-like sequence, are not required for virus replication and have no obvious effect on viral RNA transcription in the presence of an intact TATA box. However, in mutants lacking a functional TATA sequence, mutations in this region serve to decrease the efficiency of correct transcriptional initiation events.