Publications

Abstract

It is known that pre-mRNAs in eukaryotic cells can be processed to circular RNAs by a backsplicing mechanism. Circular RNAs have great stability and can sequester proteins or small RNAs to exert functions on cellular pathways. Because viruses often exploit host pathways, we explored whether the RNA genome of the cytoplasmic hepatitis C virus is processed to yield virus-derived circRNAs (vcircRNAs). Computational analyses of RNA-seq experiments predicted that the viral RNA genome is fragmented to generate hundreds of vcircRNAs. More than a dozen of them were experimentally verified by rolling-circle amplification. VcircRNAs that contained the viral internal ribosome entry site were found to be translated into proteins that displayed proviral functions. Furthermore, two highly abundant, nontranslated vcircRNAs were shown to enhance viral RNA abundance. These findings argue that novel vcircRNA molecules modulate viral amplification in cells infected by a cytoplasmic RNA virus.

View details for DOI 10.1073/pnas.2313002121

View details for PubMedID 38319965

Abstract

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester mir-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5' end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.

View details for PubMedID 31075158

Abstract

The insulin-induced gene 1 protein (Insig1) inhibits the cholesterol biosynthesis pathway by retaining transcription factor SREBP in the endoplasmic reticulum, and by causing the degradation of HMGCR, the rate-limiting enzyme in cholesterol biosynthesis. Liver-specific microRNA miR-122, on the other hand, enhances cholesterol biosynthesis by an unknown mechanism. We have found that Insig1 mRNAs are generated by alternative cleavage and polyadenylation, resulting in specific isoform mRNA species. During high cholesterol abundance, the short 1.4-kb Insig1 mRNA was found to be preferentially translated to yield Insig1 protein. Precursor molecules of miR-122 down-regulated the translation of the 1.4-kb Insig1 isoform mRNA by interfering with the usage of the promoter-proximal cleavage-polyadenylation site that gives rise to the 1.4-kb Insig1 mRNA. These findings argue that precursor miR-122 molecules modulate polyadenylation site usage in Insig1 mRNAs, resulting in down-regulation of Insig1 protein abundance. Thus, precursor microRNAs may have hitherto undetected novel functions in nuclear gene expression.

View details for PubMedID 28928276

View details for PubMedCentralID PMC5689008

Abstract

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

View details for PubMedID 28636603

View details for PubMedCentralID PMC5526112

Abstract

Viral epitranscriptomics is a newly emerging field that has identified unique roles for RNA modifications in modulating life cycles of RNA viruses. Despite the observation of a handful of modified viral RNAs five decades ago, very little was known about how these modifications regulate viral life cycles, until recently. Here we review the pro- and anti-viral effects of methyl-6-adenosine in distinct viral life cycles, the role of 2' O-methyl modifications in RNA stability and innate immune sensing, and functions of adenosine to inosine modifications in retroviral life cycles. With roles for over 100 modifications in RNA still unknown, this is a rapidly emerging field that is destined to suggest novel antiviral therapies.

View details for DOI 10.1016/j.chom.2017.05.008

View details for PubMedID 28618265

View details for PubMedCentralID PMC5555051

View details for PubMedID 28408611

View details for PubMedCentralID PMC5391445

Abstract

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

View details for PubMedID 29186115

Abstract

Many viruses encode or subvert cellular microRNAs (miRNAs) to aid in their gene expression, amplification strategies, or pathogenic signatures. miRNAs typically downregulate gene expression by binding to the 3' untranslated region of their mRNA targets. As a result, target mRNAs are translationally repressed and subsequently deadenylated and degraded. Curiously, hepatitis C virus (HCV), a member of the Flaviviridae family, recruits two molecules of liver-specific microRNA-122 (miR-122) to the 5' end of its genome. In contrast to the canonical activity of miRNAs, the interactions of miR-122 with the viral genome promote viral RNA accumulation in cultured cells and in animal models of HCV infection. Sequestration of miR-122 results in loss of viral RNA both in cell culture and in the livers of chronic HCV-infected patients. This review discusses the mechanisms by which miR-122 is thought to enhance viral RNA abundance and the consequences of miR-122-HCV interactions. We also describe preliminary findings from phase II clinical trials in patients treated with miR-122 antisense oligonucleotides.

View details for PubMedID 27578438