ABSTRACT
BACKGROUND: RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA) or micro RNA (miRNA) from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The suppression of gene expression through these mechanisms serves to regulate endogenous gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed in the context of RNAi and express either a suppressor of RNAi or their own viral miRNA. RESULTS: In this study we investigated the possibility that the HIV-1 TAR element, a hairpin structure of ~50 nucleotides found at the 5' end of the HIV viral mRNA, is recognized by the RNAi machinery and processed to yield a viral miRNA. We show that the protein Dicer, the enzyme responsible for cleaving miRNA and siRNA from longer RNA sequences, is expressed in CD4+ T-cells. Interestingly, the level of expression of Dicer in monocytes is sub-optimal, suggesting a possible role for RNAi in maintaining latency in T-cells. Using a biotin labeled TAR element we demonstrate that Dicer binds to this structure. We show that recombinant Dicer is capable of cleaving the TAR element in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts. Finally, we show that a TAR derived miRNA is capable of regulating viral gene expression and may be involved in repressing gene expression through transcriptional silencing. CONCLUSION: HIV-1 TAR element is processed by the Dicer enzyme to create a viral miRNA. This viral miRNA is detectable in infected cells and appears to contribute to viral latency.
Subject(s)
DEAD-box RNA Helicases/metabolism , Endoribonucleases/metabolism , HIV Long Terminal Repeat/genetics , HIV-1/genetics , MicroRNAs/metabolism , Blotting, Western , CD4 Antigens/metabolism , Cell Line , Cells, Cultured , Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation , DEAD-box RNA Helicases/genetics , Endoribonucleases/genetics , Gene Expression Regulation, Viral , HIV-1/growth & development , HIV-1/metabolism , Humans , Jurkat Cells , Luciferases/genetics , Luciferases/metabolism , MicroRNAs/genetics , Mutation , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribonuclease III , T-Lymphocytes/metabolism , T-Lymphocytes/virology , U937 CellsABSTRACT
Microarray profiling of RNA expression is a powerful tool that generates large lists of transcripts that are potentially relevant to a disease or treatment. However, because the lists of changed transcripts are embedded in figures and tables, they are typically inaccessible for search engines. Due to differences in gene nomenclatures, the lists are difficult to compare between studies. LOLA (Lists of Lists Annotated) is an internet-based database for comparing gene lists from microarray studies or other genomic-scale methods. It serves as a common platform to compare and reannotate heterogeneous gene lists from different microarray platforms or different genomic methodologies such as serial analysis of gene expression (SAGE) or proteomics. LOLA () provides researchers with a means to store, annotate, and compare gene lists produced from different studies or different analyses of the same study. It is especially useful in identifying potentially "high interest" genes which are reported as significant across multiple studies and species. Its application to the fields of stem cell, cancer, and aging research is demonstrated by comparing published papers.
