Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters.

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

Regulation of snRNA gene expression by the Drosophila melanogaster small nuclear RNA activating protein complex (DmSNAPc).

The small nuclear RNAs (snRNAs) are an essential class of non-coding RNAs first identified over 30 years ago. Many of the well-characterized snRNAs are involved in RNA processing events. However, it is now evident that other small RNAs, synthesized using similar mechanisms, play important roles at many stages of gene expression. The accurate and efficient control of the expression of snRNA (and related) genes is thus critical for cell survival. All snRNA genes share a very similar promoter structure, and their transcription is dependent upon the same multi-subunit transcription factor, termed the snRNA activating protein complex (SNAPc). Despite those similarities, some snRNA genes are transcribed by RNA polymerase II (Pol II), but others are transcribed by RNA polymerase III (Pol III). Thus snRNA genes provide a unique opportunity to understand how RNA polymerase specificity is determined and how distinct transcription machineries can interact with a common factor. This review will describe efforts taken toward solving those questions by using the fruit fly as a model organism. Drosophila melanogaster SNAPc (DmSNAPc) binds to a proximal sequence element (PSEA) present in both Pol II and Pol III snRNA promoters. Just a few differences in nucleotide sequence in the Pol II and Pol III PSEAs play a major role in determining RNA polymerase specificity. Furthermore, these same nucleotide differences result in alternative conformations of DmSNAPc on Pol II and Pol III snRNA gene promoters. It seems likely that these DNA-induced alternative DmSNAPc conformations are responsible for the differential recruitment of the distinct transcriptional machineries.

A map of Drosophila melanogaster small nuclear RNA-activating protein complex (DmSNAPc) domains involved in subunit assembly and DNA binding.

Transcription of genes coding for the small nuclear RNAs (snRNAs) is dependent upon a unique transcription factor known as the small nuclear RNA-activating protein complex (SNAPc). SNAPc binds to an essential proximal sequence element located about 40-65 base pairs upstream of the snRNA transcription start site. In the fruit fly Drosophila melanogaster, DmSNAPc contains three distinct polypeptides (DmSNAP190, DmSNAP50, and DmSNAP43) that are stably associated with each other and bind to the DNA as a complex. We have used mutational analysis to identify domains within each subunit that are involved in complex formation with the other two subunits in vivo. We have also identified domains in each subunit required for sequence-specific DNA binding. With one exception, domains required for subunit-subunit interactions lie in the most evolutionarily conserved regions of the proteins. However, DNA binding by DmSNAPc is dependent not only upon the conserved regions but is also highly dependent upon domains outside the conserved regions. Comparison with functional domains identified in human SNAPc indicates many parallels but also reveals significant differences in this ancient yet rapidly evolving system.

Regulation of Mammalian Gene Dosage by Long Noncoding RNAs.

Recent transcriptome studies suggest that long noncoding RNAs (lncRNAs) are key components of the mammalian genome, and their study has become a new frontier in biomedical research. In fact, lncRNAs in the mammalian genome were identified and studied at particular epigenetic loci, including imprinted loci and X-chromosome inactivation center, at least two decades ago-long before development of high throughput sequencing technology. Since then, researchers have found that lncRNAs play essential roles in various biological processes, mostly during development. Since much of our understanding of lncRNAs originates from our knowledge of these well-established lncRNAs, in this review we will focus on lncRNAs from the X-chromosome inactivation center and the Dlk1-Dio3 imprinted cluster as examples of lncRNA mechanisms functioning in the epigenetic regulation of mammalian genes.

Localization of residues in a novel DNA-binding domain of DmSNAP43 required for DmSNAPc DNA-binding activity.

Transcription of snRNA genes depends upon the recognition of the proximal sequence element (PSE) by the snRNA activating protein complex SNAPc. In Drosophila melanogaster, all subunits of DmSNAPc (DmSNAP43, DmSNAP50, and DmSNAP190) are required for PSE-binding activity. Previous work demonstrated that a non-canonical DmSNAP43 domain bounded by residues 193-272 was essential for DmSNAPc to bind to the PSE. In this study, the contribution of amino acid residues within this domain to DNA binding by DmSNAPc was investigated by alanine-scanning mutagenesis. The results have identified two clusters of residues within this domain required for the sequence-specific DNA-binding activity of DmSNAPc.

Ribavirin enhances interferon signaling via stimulation of mTOR and p53 activities.

Cellular mechanisms involving the enhancement of interferon (IFN) signaling by ribavirin remain poorly understood. Here, we identified a novel role of ribavirin in the communication between p53 and the mammalian target of rapamycin (mTOR) signaling. Ribavirin activates p53 by stimulating mTOR and promoting the interaction between mTOR and p53. Activated p53 stimulates the transcription of IFN regulatory factor 9 and subsequently enhances IFN signaling. Furthermore, ribavirin-induced activation of mTOR and p53 enhances IFN-dependent signaling for the IFN-alpha/ribavirin combined treatment. We conclude that ribavirin enhances activities of mTOR and p53, which may account for its antiviral and antitumor effects.