ABSTRACT
An iterative in vitro splicing strategy was employed to select for optimal 3' splicing signals from a pool of pre-mRNAs containing randomized regions. Selection of functional branchpoint sequences in HeLa cell nuclear extract yielded a sequence motif that evolved from UAA after one round of splicing toward a UACUAAC consensus after seven rounds. A significant part of the selected sequences contained a conserved AAUAAAG motif that proved to be functional both as a polyadenylation signal and a branch site in a competitive manner. Characterization of the branchpoint in these clones to either the upstream or downstream adenosines of the AAUAAAG sequence revealed that the branching process proceeded efficiently but quite promiscuously. Surprisingly, the conserved guanosine, adjacent to the common AAUAAA polyadenylation motif, was found to be required only for polyadenylation. In an independent experiment, sequences surrounding an optimal branchpoint sequence were selected from two randomized 20-nt regions. The clones selected after six rounds of splicing revealed an extended polypyrimidine tract with a high frequency of UCCU motifs and a highly conserved YAG sequence in the extreme 3' end of the randomized insert. Mutating the 3' terminal guanosine of the intron strongly affects complex A formation, implying that the invariant AG is recognized early in spliceosome assembly.
Subject(s)
RNA Splicing/genetics , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid/genetics , Base Sequence , Cloning, Molecular , Consensus Sequence/genetics , Exons/genetics , Guanosine/metabolism , Humans , Kinetics , Molecular Sequence Data , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Poly A/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins , Sequence Alignment , Serine-Arginine Splicing FactorsABSTRACT
Encapsidation of HIV-1 genomic RNA is mediated by specific interactions between the RNA packaging signal and the Gag protein. During maturation of the virion, the Gag protein is processed into smaller fragments, including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathione- S -transferase (GST) Gag and NC fusion proteins from HIV-1, to the entire HIV-1 and -2 leader RNAencompassing the packaging signal. We have mapped the binding sites at conditions where only about two complexes are formed and find that GST-Gag and GST-NC fusion proteins bind specifically to discrete sites within the leader. Analysis of the HIV-1 leader indicated that GST-Gag strongly associates with the PSI stem-loop and to a lesser extent with regions near the primer binding site. GST-NC binds the same regions but with reversed preferences. The HIV-1 proteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context.
Subject(s)
Gene Products, gag/metabolism , HIV-1/genetics , HIV-1/metabolism , HIV-2/genetics , HIV-2/metabolism , Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , Base Sequence , Binding Sites/genetics , DNA Primers/genetics , Gene Products, gag/chemistry , Gene Products, gag/genetics , HIV-1/physiology , HIV-2/physiology , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Peptide Mapping , Polymerase Chain Reaction , Protein Binding , RNA, Viral/chemistry , RNA, Viral/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Virus ReplicationABSTRACT
Continuous replication of human immunodeficiency virus type I (HIV-1) requires balanced expression of spliced and nonspliced mRNAs in the cytoplasm. This process is regulated post-transcriptionally by the viral-encoded Rev protein. An important prerequisite for Rev responsiveness is the presence of weak splice sites in the viral mRNA. We have investigated the splicing of the second intron of the HIV-1 Tat/Rev transcript in vitro and show that the 3'-splice site region is responsible for the inefficient splicing of the HIV-1 transcript. In contrast, the HIV-1 5'-splice site is highly functional in combination with a heterologous 3'-splice site. Incubation of the HIV-1 transcript in nuclear extract leads to a rapid accumulation of 50 S nonproductive pre-spliceosome complexes. These complexes contain mainly U1 and U2 small nuclear ribonucleoproteins and are formed independently of the presence of the downstream 3'-splice site. The HIV-1 transcripts, which do proceed through the first splicing step, utilize primarily a uridine as the branch acceptor nucleotide. Sequence comparison with other HIV-1 introns suggests that nucleotides other than adenosines are commonly used as branch points in these viruses.
