RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome wide.

RNA:DNA hybrids compromise replication fork progression and genome integrity in all cells. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate strongly in noncoding RNAs and 5'-UTRs of coding sequences. For ΔrnhB, hybrids accumulate preferentially in untranslated regions and early in coding sequences. We show that hybrid accumulation is particularly sensitive to gene expression in ΔrnhC cells. DNA replication in ΔrnhC cells is disrupted, leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts cause mutagenesis and shape genome organization.

RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome-wide.

RNA:DNA hybrids such as R-loops affect genome integrity and DNA replication fork progression. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome-wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation, but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate most strongly in non-coding RNAs and 5'-UTRs of coding sequences. For Δ rnhB , hybrids accumulate preferentially in untranslated regions and early in coding sequences. Hybrid accumulation is particularly sensitive to gene expression in Δ rnhC ; in cells lacking RnhC, DNA replication is disrupted leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts interact with replication to cause mutagenesis and shape genome organization.

Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria.

There is increasing evidence that prokaryotes maintain chromosome structure, which in turn impacts gene expression. We recently characterized densely occupied, multi-kilobase regions in the E. coli genome that are transcriptionally silent, similar to eukaryotic heterochromatin. These extended protein occupancy domains (EPODs) span genomic regions containing genes encoding metabolic pathways as well as parasitic elements such as prophages. Here, we investigate the contributions of nucleoid-associated proteins (NAPs) to the structuring of these domains, by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and B. subtilis. We identify key NAPs contributing to the silencing of specific EPODs, whose deletion opens a chromosomal region for RNA polymerase binding at genes contained within that region. We show that changes in E. coli EPODs facilitate an extra layer of transcriptional regulation, which prepares cells for exposure to exotic carbon sources. Furthermore, we distinguish novel xenogeneic silencing roles for the NAPs Fis and Hfq, with the presence of at least one being essential for cell viability in the presence of domesticated prophages. Our findings reveal previously unrecognized mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements.

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens.

BACKGROUND: The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. RESULTS: Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. CONCLUSION: We conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

Unexpected functions of tRNA and tRNA processing enzymes.

tRNA and tRNA processing enzymes impact more than protein production. Studies have uncovered roles for tRNA in the regulation of transcription, translation and protein turnover. Induced by stress or as a programmed part of development, nonrandom tRNA fragments can guide mRNA cleavage, inhibit translation and promote morphological changes. Similarly, tRNA processing enzymes, such as RNaseP and tRNA aminoacyl-synthetases participate in tasks affecting more than tRNA function (i.e., mRNA function and cellular signaling). Unraveling the complexities of their functions will increase our understanding of how mutations associated with disease impact these functions and the downstream consequences. This chapter focuses on how tRNA and tRNA processing enzymes influence cellular function and RNA-infrastructure via pathways beyond the decoding activities that tRNA are known for.

P-body components, Dhh1 and Pat1, are involved in tRNA nuclear-cytoplasmic dynamics.

The nuclear-cytoplasmic distribution of tRNA depends on the balance between tRNA nuclear export/re-export and retrograde tRNA nuclear import in Saccharomyces cerevisiae. The distribution of tRNA is sensitive to nutrient availability as cells deprived of various nutrients exhibit tRNA nuclear accumulation. Starvation induces numerous events that result in translational repression and P-body formation. This study investigated the possible coordination of these responses with tRNA nuclear-cytoplasmic distribution. Dhh1 and Pat1 function in parallel to promote translation repression and P-body formation in response to starvation. Loss of both, Dhh1 and Pat1, results in a failure to repress translation and to induce P-body formation in response to glucose starvation. This study reports that nutrient deprived dhh1 pat1 cells also fail to accumulate tRNA within nuclei. Conversely, inhibition of translation initiation and induction of P-body formation by overproduction of Dhh1 or Pat1 cause tRNA nuclear accumulation in nutrient-replete conditions. Also, loss of the mRNA decapping activator, Lsm1, causes tRNA nuclear accumulation. However, the coordination between P-body formation, translation repression, and tRNA distribution is limited to the early part of the P-body formation/translation repression pathway as loss of mRNA decapping or 5' to 3' degradation does not influence tRNA nuclear-cytoplasmic dynamics. The data provide the first link between P-body formation/translation initiation and tRNA nuclear-cytoplasmic dynamics. The current model is that Dhh1 and Pat1 function in parallel to promote starvation-induced tRNA nuclear accumulation.

Rapid and reversible nuclear accumulation of cytoplasmic tRNA in response to nutrient availability.

Cytoplasmic tRNAs have recently been found to accumulate in the nucleus during amino acid starvation in yeast. The mechanism and regulation by which tRNAs return to the nucleus are unclear. Here, we show accumulation of cytoplasmic tRNA in the nucleus also occurs during glucose starvation. Nuclear accumulation of tRNA in response to acute glucose or amino acid starvation is rapid, reversible, requires no new transcription, and is independent of the aminoacylation status of tRNA. Gradual depletion of nutrients also results in the accrual of tRNA in the nucleus. Distinct signal transduction pathways seem to be involved in the accumulation of cytoplasmic tRNA in the nucleus in response to amino acid versus glucose starvation. These findings suggest tRNA nucleocytoplasmic distribution may play a role in gene expression in response to nutritional stress.

Inorganic phosphate deprivation causes tRNA nuclear accumulation via retrograde transport in Saccharomyces cerevisiae.

Nuclear export of tRNA is an essential eukaryotic function, yet the one known yeast tRNA nuclear exporter, Los1, is nonessential. Moreover recent studies have shown that tRNAs can move retrograde from the cytosol to the nucleus by an undefined process. Therefore, additional gene products involved in tRNA nucleus-cytosol dynamics have yet to be identified. Synthetic genetic array (SGA) analysis was employed to identify proteins involved in Los1-independent tRNA transport and in regulating tRNA nucleus-cytosol distribution. These studies uncovered synthetic interactions between los1Delta and pho88Delta involved in inorganic phopsphate uptake. Further analysis revealed that inorganic phosphate deprivation causes transient, temperature-dependent nuclear accumulation of mature cytoplasmic tRNA within nuclei via a Mtr10- and retrograde-dependent pathway, providing a novel connection between tRNA subcellular dynamics and phosphate availability.

Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C.

The essential Saccharomyces cerevisiae tRNA(His) guanylyltransferase (Thg1p) is responsible for the unusual G(-1) addition to the 5' end of cytoplasmic tRNA(His). We report here that tRNA(His) from Thg1p-depleted cells is uncharged, although histidyl tRNA synthetase is active and the 3' end of the tRNA is intact, suggesting that G(-1) is a critical determinant for aminoacylation of tRNA(His) in vivo. Thg1p depletion leads to activation of the GCN4 pathway, most, but not all, of which is Gcn2p dependent, and to the accumulation of tRNA(His) in the nucleus. Surprisingly, tRNA(His) in Thg1p-depleted cells accumulates additional m(5)C modifications, which are delayed relative to the loss of G(-1) and aminoacylation. The additional modification is likely due to tRNA m(5)C methyltransferase Trm4p. We developed a new method to map m(5)C residues in RNA and localized the additional m(5)C to positions 48 and 50. This is the first documented example of the accumulation of additional modifications in a eukaryotic tRNA species.