Biology subjects

Karasik, A.

Publications and source records attributed to Karasik, A..

6 recordsLinked to original sources

bioRxiv2025

Stalled translation on transcripts cleaved by RNase L activates signaling important for innate immunity

RNase L is an endonuclease that responds to infections by cleaving most host- and pathogen-derived single-stranded RNAs. This widespread RNA cleavage can lead to death of the infected cell via the ribotoxic stress response (RSR). An ongoing challenge is to understand how RNase Ls endonuclease activity triggers cell death to benefit the host. To address this question, we used nanopore-based long-read sequencing to show that 3 mRNA fragments in the cell were not fully degraded after RNase L activation and that these fragments were translated by ribosomes. We further asked whether ribosomes on mRNA fragments stall when they reach 3 ends created by RNase L. We used ribosome profiling to capture footprints protected by these ribosomes, which can be identified by their short length (15-18 nt). We found that RNase L activation increased the number of stalled ribosomes at RNase L cleavage sites. Loss of the ribosome rescue factor PELO increased the number of short footprints derived from stalled ribosomes and augmented the RSR. Our work therefore establishes a role for fragmented mRNA in causing ribosome stalling that promotes innate immunity via the RSR. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/658914v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1f08b5forg.highwire.dtl.DTLVardef@14e2cd1org.highwire.dtl.DTLVardef@160814corg.highwire.dtl.DTLVardef@c8ddb4_HPS_FORMAT_FIGEXP M_FIG Stalled translation of mRNAs that are fragmented by RNase L leads to ribosome stalling and potentially collisions. Stalled ribosomes are rescued by PELO or activate innate immune signaling via ZAK. Renderings based on PDB 4o1o and 3jag. C_FIG HighlightsO_LIActivation of RNase L leads to accumulation and translation of mRNA fragments C_LIO_LIRibosomes stall at the 3 end of the RNase L cleaved mRNA fragments C_LIO_LIPELO rescues ribosomes stalled due to RNase L activation C_LI

molecular biology↗

bioRxiv2023

Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response

Viral infection triggers several dsRNA sensors that lead to changes in gene expression in the cell. One of these sensors activates an endonuclease, RNase L, that cleaves single stranded RNA. However, how the resultant widespread RNA fragmentation affects gene expression is not fully understood. Here we show that this fragmentation induces the Ribotoxic Stress Response via ZAK, potentially through ribosome collisions. The p38 and JNK pathways that are activated as part of this response promote outcomes that inhibit the virus, such as programmed cell death. We also show that RNase L limits the translation of stress-responsive genes, including antiviral IFIT mRNAs and GADD34 that encodes an antagonist of the Integrated Stress Response. Intriguingly, we found the activity of the generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage can evoke an antiviral program. HighlightsO_LIActivated RNase L acts with dsRNA-sensing pathways to promote cell signaling C_LIO_LIRNA fragmentation induces transcription through ZAK signaling C_LIO_LIActivation of RNase L modulates levels of eIF2 phosphorylation C_LIO_LITranslation of the GADD34 and IFIT mRNAs is inhibited by active RNase L C_LI

molecular biology↗

bioRxiv2022

Mode of inhibition of RNase P by gambogic acid and juglone

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5 end of precursor transfer RNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein (RNP) complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of protein-only RNase P that can be used as tools for studying the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization (FP) cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 M range were identified and analyzed. These compounds similarly inhibit human mtRNase P, a multi-subunit protein enzyme, and are 50-fold less potent against bacterial RNA-dependent RNase P. Biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor that targets the PRORP1-substrate complex while juglone acts as time-dependent inhibitor of PRORP1. X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. A model consistent with the kinetic data is that juglone binds to PRORP1 rapidly to form an inactive enzyme-inhibitor (EI) complex, and then undergoes a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

biochemistry↗

bioRxiv2020

Activation of the antiviral factor RNase L triggers translation of non-coding mRNA sequences

Ribonuclease L (RNase L) is activated as part of the innate immune response and plays an important role in the clearance of viral infections. When activated, it endonucleolytically cleaves both viral and host RNAs, leading to a global reduction in protein synthesis. However, it remains unknown how widespread RNA decay, and consequent changes in the translatome, promote the elimination of viruses. To study how this altered transcriptome is translated, we assayed the global distribution of ribosomes in RNase L activated human cells with ribosome profiling. We found that RNase L activation leads to a substantial increase in the fraction of translating ribosomes in ORFs internal to coding sequences (iORFs) and ORFs within 5 and 3 UTRs (uORFs and dORFs). Translation of these alternative ORFs was dependent on RNase Ls cleavage activity, suggesting that mRNA decay fragments are translated to produce short peptides that may be important for antiviral activity.

molecular biology↗

bioRxiv2020

Disease associated mutations in mitochondrial precursor tRNAs affect binding, m1R9 methylation and tRNA processing by mtRNase P

Mitochondrial diseases linked to mutations in mitochondrial (mt)tRNA sequences are abundant. However, the contributions of these tRNA mutations to the development of diseases is mostly unknown. Mutations may affect interactions with (mt)tRNA maturation enzymes or protein synthesis machinery leading to mitochondrial dysfunction. In human mitochondria, the first step of tRNA processing is the removal of the 5'end of precursor tRNAs (pre-tRNA) catalyzed by the three-component enzyme, mtRNase P. Additionally, one sub-component of mtRNase P, mitochondrial RNase P protein 1 (MRPP1) catalyzes methylation of the R9 base in pre-tRNAs. Despite the central role of 5' end processing in mitochondrial tRNA maturation, the link between mtRNase P and diseases is mostly unexplored. Here we investigate how 11 different human disease-linked mutations in (mt)pre-tRNAs affect the activities of mtRNase P. We find that several mutations weaken the pre-tRNA binding affinity, while the majority of mutations decrease 5' end processing and methylation activity catalyzed by mtRNase P. Furthermore, all of the investigated mutations in (mt)pre-tRNALeu(UUR) alter the tRNA fold which contributes to the partial loss of function of mtRNase P. Overall, these results reveal an etiological link between early steps of (mt)tRNA-substrate processing and mitochondrial disease.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2018

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

Human mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5 end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

biochemistry↗