RESUMO
During the early phase of adenovirus infection, the promoter-proximal L1 poly(A) site in the major late transcription unit is used preferentially despite the fact that the distal L3 poly(A) site is stronger (i.e. it competes better for processing factors and is cleaved at a faster rate, in vitro). Previous work had established that this was due at least in part to the stable binding of the processing factor, cleavage and polyadenylation specificity factor, to the L1 poly(A) site as mediated by specific regulatory sequences. It is now demonstrated that in addition, the L1 poly(A) site has a positional advantage because of its 5' location in the transcription unit. We also show that preferential processing of a particular poly(A) site in a complex transcription unit is dependent on RNA polymerase II. Our results are consistent with recent reports demonstrating that the processing factors cleavage and polyadenylation specificity factor and cleavage stimulatory factor are associated with the RNA polymerase II holoenzyme; thus, processing at a weak poly(A) site like L1 can be enhanced by virtue of its being the first site to be transcribed.
Assuntos
Adenoviridae/genética , RNA Polimerase II/fisiologia , RNA Mensageiro/metabolismo , Transcrição Gênica , Células Cultivadas , RNA Polimerases Dirigidas por DNA/fisiologia , Células HeLa , Humanos , Regiões Promotoras Genéticas , Precursores de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Proteínas ViraisRESUMO
The adenovirus major late transcription unit encodes five poly(A) sites whose use during infection is regulated. Early in the infection, the 5'-most site, L1, is used preferentially, whereas late in infection, all sites are used equivalently. Previous in vivo experiments indicated that regulatory sequences flank the AAUAAA and GU-rich elements of the L1 poly(A) site. We have developed an in vitro coupled transcription-processing system for studying the function of these regulatory sequences in HeLa cell nuclear extracts. The in vitro analysis using this system shows that predominant use of the L1 poly(A) site, as mediated by the upstream regulatory sequence, is independent of transcription. Furthermore, the reaction conditions are favorable to both 3'-end processing and splicing, making this system generally useful for the study of posttranscriptional processes.
Assuntos
Adenoviridae/metabolismo , Núcleo Celular/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Sequências Reguladoras de Ácido Nucleico , Adenoviridae/genética , Northern Blotting , Sistema Livre de Células , Células HeLa , Humanos , Hibridização de Ácido Nucleico , Poli A/biossíntese , Splicing de RNA , RNA Mensageiro/biossínteseRESUMO
In retroviral proviruses, the poly(A) site is present in both long terminal repeats (LTRs) but used only in the 3' position. One mechanism to account for this selective poly(A) site usage is that LTR U3 sequences, transcribed only from the 3' poly(A) site, are required in the RNA for efficient processing. To test this possibility, mutations were made in the human immunodeficiency virus type 1 (HIV-1) U3 region and the mutated LTRs were inserted into simple and complex transcription units. HIV-1 poly(A) site usage was then quantitated by S1 nuclease analysis following transfection of each construct into human 293 cells. The results showed that U3 sequences confined to the transcription control region were required for efficient usage of the HIV-1 poly(A) site, even when it was placed 1.5 kb from the promoter. Although the roles of U3 in processing and transcription activation were separable, optimal 3' end formation was partly dependent on HIV-1 enhancer and SP1 binding site sequences.
