tRNA Dysregulation in Neurodevelopmental and Neurodegenerative Diseases.

Transfer RNAs (tRNAs) decode messenger RNA codons to peptides at the ribosome. The nuclear genome contains many tRNA genes for each amino acid and even each anticodon. Recent evidence indicates that expression of these tRNAs in neurons is regulated, and they are not functionally redundant. When specific tRNA genes are nonfunctional, this results in an imbalance between codon demand and tRNA availability. Furthermore, tRNAs are spliced, processed, and posttranscriptionally modified. Defects in these processes lead to neurological disorders. Finally, mutations in the aminoacyl tRNA synthetases (aaRSs) also lead to disease. Recessive mutations in several aaRSs cause syndromic disorders, while dominant mutations in a subset of aaRSs lead to peripheral neuropathy, again due to an imbalance between tRNA supply and codon demand. While it is clear that disrupting tRNA biology often leads to neurological disease, additional research is needed to understand the sensitivity of neurons to these changes.

Signaling to and from the RNA Polymerase III Transcription and Processing Machinery.

RNA polymerase (Pol) III has a specialized role in transcribing the most abundant RNAs in eukaryotic cells, transfer RNAs (tRNAs), along with other ubiquitous small noncoding RNAs, many of which have functions related to the ribosome and protein synthesis. The high energetic cost of producing these RNAs and their central role in protein synthesis underlie the robust regulation of Pol III transcription in response to nutrients and stress by growth regulatory pathways. Downstream of Pol III, signaling impacts posttranscriptional processes affecting tRNA function in translation and tRNA cleavage into smaller fragments that are increasingly attributed with novel cellular activities. In this review, we consider how nutrients and stress control Pol III transcription via its factors and its negative regulator, Maf1. We highlight recent work showing that the composition of the tRNA population and the function of individual tRNAs is dynamically controlled and that unrestrained Pol III transcription can reprogram central metabolic pathways.

The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

ALKBH1-Mediated tRNA Demethylation Regulates Translation.

tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.

RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

Transcriptome-wide Mapping of Internal N7-Methylguanosine Methylome in Mammalian mRNA.

N7-methylguanosine (m7G) is a positively charged, essential modification at the 5' cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m7G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m7G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m7G methylome using m7G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m7G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m7G-seq enabled transcriptome-wide mapping of m7G in human tRNA and mRNA, revealing distribution features of the internal m7G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m7G within mRNA and showed that internal m7G methylation could affect mRNA translation.

tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen Pseudomonas aeruginosa.

The success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered, and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that the enzymatic activity of GidA leads to the introduction of a carboxymethylaminomethyl modification in selected tRNAs. Modifications at the wobble uridine base (cmnm5U34) of the anticodon drives translation of transcripts containing rare codons. Specifically, in P. aeruginosa the presence of GidA-dependent tRNA modifications modulates expression of genes encoding virulence regulators, leading to a cellular proteomic shift toward pathogenic and well-adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept. It provides a paradigm of how environmentally driven tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to challenging habitats prevailing in the host niche.

Modifications in the T arm of tRNA globally determine tRNA maturation, function, and cellular fitness.

Almost all elongator tRNAs (Transfer RNAs) harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, generated by the enzymes TrmA and TruB, respectively, in Escherichia coli. TrmA and TruB both act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wild type. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modifications such as acp3U47. While overall protein synthesis is not affected in ΔtrmA and ΔtruB strains, the translation of a subset of codons is significantly impaired. As a consequence, we observe translationally reduced expression of many specific proteins, that are either encoded with a high frequency of these codons or that are large proteins. The resulting proteome changes are not related to a specific growth phenotype, but overall cellular fitness is impaired upon deleting trmA and truB in accordance with a general protein synthesis impact. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness irrespective of the growth conditions which explains the conservation of trmA and truB.

tRNAVal allows four-way decoding with unmodified uridine at the wobble position in Lactobacillus casei.

Modifications at the wobble position (position 34) of tRNA facilitate interactions that enable or stabilize non-Watson-Crick basepairs. In bacterial tRNA, 5-hydroxyuridine (ho5U) derivatives xo5U [x: methyl (mo5U), carboxymethyl (cmo5U), and methoxycarbonylmethyl (mcmo5U)] present at the wobble positions of tRNAs are responsible for recognition of NYN codon families. These modifications of U34 allow basepairing not only with A and G but also with U and in some cases C. mo5U was originally found in Gram-positive bacteria, and cmo5U and mcmo5U were found in Gram-negative bacteria. tRNAs of Mycoplasma species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. Lactobacillus casei, Gram-positive bacteria and lactic acid bacteria, lacks the modification enzyme genes for xo5U biosynthesis. Nevertheless, L. casei has only one type of tRNAVal with the anticodon UAC [tRNAVal(UAC)]. However, the genome of L. casei encodes an undetermined tRNA (tRNAUnd) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in L. casei tRNAVal is unmodified and that there is no tRNAUnd expression in the cells. In addition, in vitro transcribed tRNAUnd was not aminoacylated by L. casei valyl-tRNA synthetase suggesting that tRNAUnd is not able to accept valine, even if expressed in cells. Correspondingly, native tRNAVal(UAC) with unmodified U34 bound to all four valine codons in the ribosome A site. This suggests that L. casei tRNAVal decodes all valine codons by four-way decoding, similarly to tRNAs from Mycoplasma species, mitochondria, and chloroplasts.

A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a single homolog of the tRNA methyltransferase Trm10 performs m1G9 modification on 13 different tRNAs. Here we provide evidence that the m1G9 modification catalyzed by S. cerevisiae Trm10 plays a biologically important role for one of these tRNA substrates, tRNATrp Overexpression of tRNATrp (and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of the trm10Δ strain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNATrp is depleted in trm10Δ cells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNAGly) is not affected under these conditions. Thus, m1G9 in S. cerevisiae is another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m1G9 on mature tRNATrp, precursor tRNATrp species accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNATrp are rescued in the trm10Δmet22Δ strain, consistent with the known role of Met22 in tRNA quality control, where deletion of met22 causes inhibition of 5'-3' exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for the decay of hypomodified tRNATrp, based on the inability of mutants of each enzyme to rescue the growth of the trm10Δ strain in the presence of 5FU. Thus, the surveillance of tRNATrp appears to constitute a distinct tRNA quality control pathway in S. cerevisiae.

The modification landscape of Pseudomonas aeruginosa tRNAs.

RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here, we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in Escherichia coli, which enabled us to identify similar modifications in P. aeruginosa Our analysis supports a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA-modifying enzymes, some of which play roles in determining virulence and pathogenicity.

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNAGlu, Gln, Asp contains a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm5s2U to mnm5s2U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Phosphate Limitation Responses in Marine Green Algae Are Linked to Reprogramming of the tRNA Epitranscriptome and Codon Usage Bias.

Marine algae are central to global carbon fixation, and their productivity is dictated largely by resource availability. Reduced nutrient availability is predicted for vast oceanic regions as an outcome of climate change; however, there is much to learn regarding response mechanisms of the tiny picoplankton that thrive in these environments, especially eukaryotic phytoplankton. Here, we investigate responses of the picoeukaryote Micromonas commoda, a green alga found throughout subtropical and tropical oceans. Under shifting phosphate availability scenarios, transcriptomic analyses revealed altered expression of transfer RNA modification enzymes and biased codon usage of transcripts more abundant during phosphate-limiting versus phosphate-replete conditions, consistent with the role of transfer RNA modifications in regulating codon recognition. To associate the observed shift in the expression of the transfer RNA modification enzyme complement with the transfer RNAs encoded by M. commoda, we also determined the transfer RNA repertoire of this alga revealing potential targets of the modification enzymes. Codon usage bias was particularly pronounced in transcripts encoding proteins with direct roles in managing phosphate limitation and photosystem-associated proteins that have ill-characterized putative functions in "light stress." The observed codon usage bias corresponds to a proposed stress response mechanism in which the interplay between stress-induced changes in transfer RNA modifications and skewed codon usage in certain essential response genes drives preferential translation of the encoded proteins. Collectively, we expose a potential underlying mechanism for achieving growth under enhanced nutrient limitation that extends beyond the catalog of up- or downregulated protein-encoding genes to the cell biological controls that underpin acclimation to changing environmental conditions.

Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.

High-resolution crystal structure of RNA kinase ArK1 from G. acetivorans.

U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.

Queuosine salvage in Bartonella henselae Houston 1: a unique evolutionary path.

Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: (1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and (2) queuosine precursor transporter (QPTR), a transporter protein that imports Q precursors. Organisms such as the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, MS analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ1, akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens - from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ1 confers fitness advantages when B. henselae is growing outside a mammalian host.

The first Turkish family with a novel biallelic missense variant of the ALKBH8 gene: A study on the clinical and variant spectrum of ALKBH8-related intellectual developmental disorders.

ABH8, the protein encoded by the ALKBH8 gene, modifies tRNAs by methylating their anticodon wobble uridine residues. The variations in the ALKBH8 gene are associated with the "intellectual developmental disorder, autosomal recessive type 71" (MIM: 618504) phenotype in the OMIM database. This phenotype is characterized by global developmental delay, facial dysmorphic features, and psychiatric problems. To date, 12 patients from five distinct families carrying variants of the ALKBH8 gene have been reported in the literature. In the present study, we report the first Turkish family harboring a novel homozygous missense variant, NM_138775.3:c.1874G > C (p.Arg625Pro), in the last exon of the ALKBH8 gene. Two affected siblings in this family showed signs of global developmental delay and intellectual disability. Based on the dysmorphological assessment of the cases, fifth finger clinodactyly and fetal fingertip pads were prominent, in addition to the dysmorphic findings similar to those reported in previous studies. Minor dysmorphic limb anomalies in relation to this phenotype have not yet been previously reported in the literature. Our computational studies revealed the potential deleterious effects of the Arg-to-Pro substitution on the structure and stability of the ABH8 methyltransferase domain. In the present report, the first Turkish family with an ultrarare disease associated with the ALKBH8 gene was reported, and a novel deleterious variant in the ALKBH8 gene and additional clinical features that were not reported with this condition have been reported.

tRNA modifications and tRNA-derived small RNAs: new insights of tRNA in human disease.

tRNAs are codon decoders that convert the transcriptome into the proteome. The field of tRNA research is excited by the increasing discovery of specific tRNA modifications that are installed at specific, evolutionarily conserved positions by a set of specialized tRNA-modifying enzymes and the biogenesis of tRNA-derived regulatory fragments (tsRNAs) which exhibit copious activities through multiple mechanisms. Dysregulation of tRNA modification usually has pathological consequences, a phenomenon referred to as "tRNA modopathy". Current evidence suggests that certain tRNA-modifying enzymes and tsRNAs may serve as promising diagnostic biomarkers and therapeutic targets, particularly for chemoresistant cancers. In this review, we discuss the latest discoveries that elucidate the molecular mechanisms underlying the functions of clinically relevant tRNA modifications and tsRNAs, with a focus on malignancies. We also discuss the therapeutic potential of tRNA/tsRNA-based therapies, aiming to provide insights for the development of innovative therapeutic strategies. Further efforts to unravel the complexities inherent in tRNA biology hold the promise of yielding better biomarkers for the diagnosis and prognosis of diseases, thereby advancing the development of precision medicine for health improvement.

O-Sialoglycoprotein Endopeptidase Deficiency Impairs Proteostasis and Induces Autophagy in Human Embryonic Stem Cells.

The OSGEP gene encodes O-sialoglycoprotein endopeptidase, a catalytic unit of the highly conserved KEOPS complex (Kinase, Endopeptidase, and Other Proteins of small Size) that regulates the second biosynthetic step in the formation of N-6-threonylcarbamoyladenosine (t6A). Mutations in KEOPS cause Galloway-Mowat syndrome (GAMOS), whose cellular function in mammals and underlying molecular mechanisms are not well understood. In this study, we utilized lentivirus-mediated OSGEP knockdown to generate OSGEP-deficient human embryonic stem cells (hESCs). OSGEP-knockdown hESCs exhibited reduced stemness factor expression and G2/M phase arrest, indicating a potential role of OSGEP in the regulation of hESC fate. Additionally, OSGEP silencing led to enhanced protein synthesis and increased aggregation of proteins, which further induced inappropriate autophagy, as evidenced by the altered expression of P62 and the conversion of LC3-I to LC3-II. The above findings shed light on the potential involvement of OSGEP in regulating pluripotency and differentiation in hESCs while simultaneously highlighting its crucial role in maintaining proteostasis and autophagy, which may have implications for human disease.

Synthetic lethal mutants in Escherichia coli define pathways necessary for survival with RNase H deficiency.

Ribonucleotides frequently contaminate DNA and, if not removed, cause genomic instability. Consequently, all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids (RDHs). Escherichia coli lacking RNase HI (rnhA) and RNase HII (rnhB) enzymes, the ∆rnhA ∆rnhB double mutant, accumulates RDHs in its DNA. These RDHs can convert into RNA-containing DNA lesions (R-lesions) of unclear nature that compromise genomic stability. The ∆rnhAB double mutant has severe phenotypes, like growth inhibition, replication stress, sensitivity to ultraviolet radiation, SOS induction, increased chromosomal fragmentation, and defects in nucleoid organization. In this study, we found that RNase HI deficiency also alters wild-type levels of DNA supercoiling. Despite these severe chromosomal complications, ∆rnhAB double mutant survives, suggesting that dedicated pathways operate to avoid or repair R-lesions. To identify these pathways, we systematically searched for mutants synthetic lethal (colethal) with the rnhAB defect using an unbiased color screen and a candidate gene approach. We identified both novel and previously reported rnhAB-colethal and -coinhibited mutants, characterized them, and sorted them into avoidance or repair pathways. These mutants operate in various parts of nucleic acid metabolism, including replication fork progression, R-loop prevention and removal, nucleoid organization, tRNA modification, recombinational repair, and chromosome-dimer resolution, demonstrating the pleiotropic nature of RNase H deficiency. IMPORTANCE Ribonucleotides (rNs) are structurally very similar to deoxyribonucleotides. Consequently, rN contamination of DNA is common and pervasive across all domains of life. Failure to remove rNs from DNA has severe consequences, and all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids. RNase H deficiency leads to complications in bacteria, yeast, and mouse, and diseases like progressive external ophthalmoplegia (mitochondrial defects in RNASEH1) and Aicardi-Goutières syndrome (defects in RNASEH2) in humans. Escherichia coli ∆rnhAB mutant, deficient in RNases H, has severe chromosomal complications. Despite substantial problems, nearly half of the mutant population survives. We have identified novel and previously confirmed pathways in various parts of nucleic acid metabolism that ensure survival with RNase H deficiency.