tRNATyr has an unusually short half-life in Trypanosoma brucei.

Following transcription, tRNAs undergo a series of processing and modification events to become functional adaptors in protein synthesis. Eukaryotes have also evolved intracellular transport systems whereby nucleus-encoded tRNAs may travel out and into the nucleus. In trypanosomes, nearly all tRNAs are also imported from the cytoplasm into the mitochondrion, which lacks tRNA genes. Differential subcellular localization of the cytoplasmic splicing machinery and a nuclear enzyme responsible for queuosine modification at the anticodon "wobble" position appear to be important quality control mechanisms for tRNATyr, the only intron-containing tRNA in T. brucei Since tRNA-guanine transglycosylase (TGT), the enzyme responsible for Q formation, cannot act on an intron-containing tRNA, retrograde nuclear transport is an essential step in maturation. Unlike maturation/processing pathways, the general mechanisms of tRNA stabilization and degradation in T. brucei are poorly understood. Using a combination of cellular and molecular approaches, we show that tRNATyr has an unusually short half-life. tRNATyr, and in addition tRNAAsp, also show the presence of slow-migrating bands during electrophoresis; we term these conformers: alt-tRNATyr and alt-tRNAAsp, respectively. Although we do not know the chemical or structural nature of these conformers, alt-tRNATyr has a short half-life resembling that of tRNATyr; the same is not true for alt-tRNAAsp We also show that RRP44, which is usually an exosome subunit in other organisms, is involved in tRNA degradation of the only intron-containing tRNA in T. brucei and is partly responsible for its unusually short half-life.

RNA binding by ADAR3 inhibits adenosine-to-inosine editing and promotes expression of immune response protein MAVS.

Members of the ADAR family of double-stranded RNA-binding proteins regulate one of the most abundant RNA modifications in humans, the deamination of adenosine to inosine. Several transcriptome-wide studies have been carried out to identify RNA targets of the active deaminases ADAR1 and ADAR2. However, our understanding of ADAR3, the brain-specific deaminase-deficient ADAR family member, is limited to a few transcripts. In this study, we identified over 3300 transcripts bound by ADAR3 and observed that binding of ADAR3 correlated with reduced editing of over 400 sites in the glioblastoma transcriptome. We further investigated the impact of ADAR3 on gene regulation of the transcript that encodes MAVS, an essential protein in the innate immune response pathway. We observed reduced editing in the MAVS 3' UTR in cells expressing increased ADAR3 or reduced ADAR1 suggesting ADAR3 acts as a negative regulator of ADAR1-mediated editing. While neither ADAR1 knockdown or ADAR3 overexpression affected MAVS mRNA expression, we demonstrate increased ADAR3 expression resulted in upregulation of MAVS protein expression. In addition, we created a novel genetic mutant of ADAR3 that exhibited enhanced RNA binding and MAVS upregulation compared with wildtype ADAR3. Interestingly, this ADAR3 mutant no longer repressed RNA editing, suggesting ADAR3 has a unique regulatory role beyond altering editing levels. Altogether, this study provides the first global view of ADAR3-bound RNAs in glioblastoma cells and identifies both a role for ADAR3 in repressing ADAR1-mediated editing and an RNA-binding dependent function of ADAR3 in regulating MAVS expression.

A protein-protein interaction underlies the molecular basis for substrate recognition by an adenosine-to-inosine RNA-editing enzyme.

Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine within double-stranded regions of RNA, resulting in increased transcriptomic diversity, as well as protection of cellular double-stranded RNA (dsRNA) from silencing and improper immune activation. The presence of dsRNA-binding domains (dsRBDs) in all ADARs suggests these domains are important for substrate recognition; however, the role of dsRBDs in vivo remains largely unknown. Herein, our studies indicate the Caenorhabditis elegans ADAR enzyme, ADR-2, has low affinity for dsRNA, but interacts with ADR-1, an editing-deficient member of the ADAR family, which has a 100-fold higher affinity for dsRNA. ADR-1 uses one dsRBD to physically interact with ADR-2 and a second dsRBD to bind to dsRNAs, thereby tethering ADR-2 to substrates. ADR-2 interacts with >1200 transcripts in vivo, and ADR-1 is required for 80% of these interactions. Our results identify a novel mode of substrate recognition for ADAR enzymes and indicate that protein-protein interactions can guide substrate recognition for RNA editors.

Gammaherpesvirus infection triggers the formation of tRNA fragments from premature tRNAs.

Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. In addition, mounting evidence supports biological function for tRNA cleavage products, including in the control of gene expression during conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to enhanced tRNA transcription. However, whether this has any influence on tRNA transcript processing, viral replication, or the host response is not known. Here, we combined two new approaches, sequencing library preparation by Ordered Two Template Relay (OTTR) and tRNA bioinformatic analysis by tRAX, to quantitatively profile full-length tRNAs and tRNA fragment (tRF) identities during MHV68 infection. We find that MHV68 infection triggers both pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tRFs. OTTR-tRAX revealed not only host tRNAome changes, but also the expression patterns of virally-encoded tRNAs (virtRNAs) and virtRFs made from the MHV68 genome, including their base modification signatures. Because the transcript ends of several host tRFs matched tRNA splice junctions, we tested and confirmed the role of tRNA splicing factors TSEN2 and CLP1 in MHV68-induced tRF biogenesis. Further, we show that CLP1 kinase, and by extension tRNA splicing, is required for productive MHV68 infection. Our findings provide new insight into how gammaherpesvirus infection both impacts and relies on tRNA transcription and processing.

Gammaherpesvirus infection alters transfer RNA splicing and triggers tRNA cleavage.

Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. Moreover, mounting evidence supports a noncanonical role for tRNA cleavage products in the control of gene expression during diverse conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to altered tRNA transcription, suggesting that tRNA regulation may play an important role in mediating viral replication or the host response. To better understand how viral infection alters tRNA expression, we combined Ordered Two Template Relay (OTTR) with tRNA-specific bioinformatic software called tRAX to profile full-length tRNAs and fragmented tRNA-derived RNAs (tDRs) during infection with MHV68. We find that OTTR-tRAX is a powerful sequencing strategy for combined tRNA/tDR profiling and reveals that MHV68 infection triggers pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tDRs. Fragments of virally-encoded tRNAs (virtRNAs), as well as virtRNA base modification signatures are also detectable during infection. We present evidence that tRNA splicing factors are involved in the biogenesis of MHV68-induced cleavage products from pre-tRNAs and, in the case of CLP1 kinase, impact infectious virus production. Our data offers new insights into the importance of tRNA processing during gammaherpesvirus infection.

Distinct Stress-Dependent Signatures of Cellular and Extracellular tRNA-Derived Small RNAs.

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB-facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress-modulated abundance of sub-populations of cellular and extracellular tDRs. Finally, a sub-population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress-responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

Disruption in A-to-I Editing Levels Affects C. elegans Development More Than a Complete Lack of Editing.

A-to-I RNA editing, catalyzed by ADAR proteins, is widespread in eukaryotic transcriptomes. Studies showed that, in C. elegans, ADR-2 can actively deaminate dsRNA, whereas ADR-1 cannot. Therefore, we set out to study the effect of each of the ADAR genes on the RNA editing process. We performed comprehensive phenotypic, transcriptomics, proteomics, and RNA binding screens on worms mutated in a single ADAR gene. We found that ADR-1 mutants exhibit more-severe phenotypes than ADR-2, and some of them are a result of non-editing functions of ADR-1. We also show that ADR-1 significantly binds edited genes and regulates mRNA expression, whereas the effect on protein levels is minor. In addition, ADR-1 primarily promotes editing by ADR-2 at the L4 stage of development. Our results suggest that ADR-1 has a significant role in the RNA editing process and in altering editing levels that affect RNA expression; loss of ADR-1 results in severe phenotypes.

The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis.

ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression.