Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNA

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNAs

Mature and functional viral miRNAs transcribed from novel RNA polymerase III promoters

Murid herpesvirus 4 (MuHV-4) microRNAs were previously cloned from latently infected tumor cells and predicted to be processed from a series of RNA polymerase III primary transcripts. We detected maturely processed MuHV-4 miRNAs within total RNA from lytically infected cells in vitro and infected tissues ex vivo, using a highly sensitive reverse ligation meditated RT-PCR strategy. We determined that the MuHV-4 microRNAs are biologically active during infection by a luciferase reporter system. We experimentally demonstrated that transcription of the MuHV-4 microRNAs is by RNA polymerase III by α-amanitin insensitivity and by specific deletion of the RNA polymerase III type 2-like promoter elements of MuHV-4, resulting in the complete loss of miRNA detection and function. Finally, we demonstrate that these 10 viral miRNAs, each transcribed from highly conserved and novel polymerase III promoter elements, vary markedly in their relative abundance and activity

Beyond the Ribosome: Extra-translational Functions of tRNA Fragments

High-throughput sequencing studies of small RNAs reveal a complex milieu of noncoding RNAs in biological samples. Early data analysis was often limited to microRNAs due to their regulatory nature and potential as biomarkers; however, many more classes of noncoding RNAs are now being recognized. A class of fragments initially excluded from analysis were those derived from transfer RNAs (tRNAs) because they were thought to be degradation products. More recently, critical cellular function has been attributed to tRNA fragments (tRFs), and their conservation across all domains of life has propelled them into an emerging area of scientific study. The biogenesis of tRFs is currently being elucidated, and initial studies show that a diverse array of tRFs are genera ted from all parts of a tRNA molecule. The goal of this review was to present what is currently known about tRFs and their potential as biomarkers for the earlier detection of disease

Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication

AbstractTo determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3′ noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3′NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3