ABSTRACT
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.
Subject(s)
HIV-1/genetics , Poly A/chemistry , Base Sequence , Electrophoresis, Polyacrylamide Gel , Fungal Proteins/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Nucleic Acid Conformation , RNA Precursors/chemistry , RNA, Viral/chemistry , RNA-Binding Proteins/metabolism , mRNA Cleavage and Polyadenylation FactorsABSTRACT
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.
Subject(s)
Enhancer Elements, Genetic , RNA, Messenger/chemistry , RNA-Binding Proteins/metabolism , Adenoviridae/genetics , Base Sequence , HIV-1/genetics , Humans , Infectious Anemia Virus, Equine/genetics , Molecular Sequence Data , Protein Binding , RNA, Messenger/metabolism , Structure-Activity Relationship , Substrate Specificity , mRNA Cleavage and Polyadenylation FactorsABSTRACT
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.
Subject(s)
Adenoviruses, Human/genetics , Poly A/metabolism , RNA Processing, Post-Transcriptional , RNA, Viral/metabolism , Adenoviruses, Human/metabolism , Animals , Cell Line , DNA-Binding Proteins/genetics , HeLa Cells , Humans , Orthohepadnavirus/genetics , RNA Precursors/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Recombination, Genetic , Sciuridae/virology , Transcription Factors/genetics , Viral Proteins , mRNA Cleavage and Polyadenylation FactorsABSTRACT
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.
Subject(s)
Enhancer Elements, Genetic/genetics , HIV-1/genetics , RNA Processing, Post-Transcriptional , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , HeLa Cells , Humans , Poly A/biosynthesis , Protein Binding , RNA Precursors/metabolism , RNA, Messenger/biosynthesis , mRNA Cleavage and Polyadenylation FactorsABSTRACT
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.
Subject(s)
HIV Long Terminal Repeat , HIV-1/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Viral/metabolism , HeLa Cells , Humans , Nucleic Acid Conformation , Poly A/metabolism , RNA, Viral/chemistryABSTRACT
Carlson's developmental theory of self-concept provides a theoretical explanation for equivalent levels of self-esteem among both sexes, despite sex differences in self-concept. The present study tests the applicability of Carlson's theory for a sample of gifted and talented female adolescents by examining three dimensions of possible self-esteem antecedents: actual talent ratings, self-perceptions of talent, and personality attributes. According to Carlson, talent ratings, self-perceptions, and personality attributes consistent with the feminine gender-role stereotype and a social orientation should emerge as positive predictors of the female adolescent's social self-esteem. Results of the regression analyses indicate that the best prediction of the social self-esteem of gifted and talented female adolescents is obtained from a combination of stereotypic feminine socially oriented and stereotypic masculine personally oriented predictor variables. For this sample, constructs such as androgyny appear to be more relevant to the understanding of social self-esteem than dichotomies such as personal-social orientation.
