Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the \'critical region\' of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

Ribosomes are large macromolecular complexes composed of both proteins and RNA, that require a plethora of factors and post-transcriptional modifications for their biogenesis. In human mitochondria, the ribosomal RNA is post-transcriptionally modified at ten sites. The N4-methylcytidine (m4C) methyltransferase, METTL15, modifies the 12S rRNA of the small subunit at position C1486. The enzyme is essential for mitochondrial protein synthesis and assembly of the mitoribosome small subunit, as shown here and by previous studies. Here, we demonstrate that the m4C modification is not required for small subunit biogenesis, indicating that the chaperone-like activity of the METTL15 protein itself is an essential component for mitoribosome biogenesis.

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3\' end was fastest when the 3\' terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

BACKGROUND: The genus Corynorhinus is composed of four recognized species: C. rafinesquii, C. townsendii, C. mexicanus, and C. leonpaniaguae, the latter two being endemic to Mexico. According to the IUCN, C. mexicanus is considered \"Near Threatened\", as its populations are dwindling and habitats are affected by anthropogenic disturbance. Corynorhinus leonpaniaguae has not been assigned to an IUCN Red List risk category due to its recent description.
RESULTS: In this study, the mitochondrial genomes of C. mexicanus and C. leonpaniaguae were assembled and characterized in detail. The mitochondrial genomes (mtDNA) of C. mexicanus and C. leonpaniaguae have lengths of 16,470 and 16,581 bp respectively, with a predominant nucleotide usage of adenine (31.670% and 31.729%, respectively) and thymine (26.15% and 26.18%, respectively). The mtDNA of C. mexicanus and C. leonpaniaguae is composed of 37 coding and non-coding elements: 22 transfer RNAs (tRNA), 13 protein-coding genes (PCGs), two ribosomal RNAs and a non-coding region, the control region, which has a length of 933 bp and 1,149 bp, respectively. All tRNAs exhibited a cloverleaf secondary structure, with the exception of trn-Ser1 which showed a deletion of the dihydrouridine arm in the two species. All PCGs are subjected to purifying selection, with atp8 being the gene showing the highest Ka/Ks value.
CONCLUSIONS: These are the first whole mitogenomic resources developed for C. mexicanus and C. leonpaniaguae and enhance our knowledge of the ecology of these species and aid in their conservation.

The RNA tailing machinery adds nucleotides to the 3\'-end of RNA molecules that are implicated in various biochemical functions, including protein synthesis and RNA stability. Here, we report a role for the RNA tailing machinery as enzymatic modifiers of intracellular amyloidogenesis. A targeted RNA interference screen identified Terminal Nucleotidyl-transferase 4b (TENT4b/Papd5) as an essential participant in the amyloidogenic phase transition of nucleoli into solid-like Amyloid bodies. Full-length-and-mRNA sequencing uncovered starRNA, a class of unusually long untemplated RNA molecules synthesized by TENT4b. StarRNA consists of short rRNA fragments linked to long, linear mixed tails that operate as polyanionic stimulators of amyloidogenesis in cells and in vitro. Ribosomal intergenic spacer noncoding RNA (rIGSRNA) recruit TENT4b in intranucleolar foci to coordinate starRNA synthesis driving their amyloidogenic phase transition. The exoribonuclease RNA Exosome degrades starRNA and functions as a general suppressor of cellular amyloidogenesis. We propose that amyloidogenic phase transition is under tight enzymatic control by the RNA tailing and exosome axis.

BACKGROUND: Ribosome biogenesis is excessively activated in tumor cells, yet it is little known whether oncogenic transcription factors (TFs) are involved in the ribosomal RNA (rRNA) transactivation.
METHODS: Nucleolar proteomics data and large-scale immunofluorescence were re-analyzed to jointly identify the proteins localized at nucleolus. RNA-Seq data of five prostate cancer (PCa) cohorts were combined and integrated with multi-dimensional data to define the upregulated nucleolar TFs in PCa tissues. Then, ChIP-Seq data of PCa cell lines and two PCa clinical cohorts were re-analyzed to reveal the TF binding patterns at ribosomal DNA (rDNA) repeats. The TF binding at rDNA was validated by ChIP-qPCR. The effect of the TF on rRNA transcription was determined by rDNA luciferase reporter, nascent RNA synthesis, and global protein translation assays.
RESULTS: In this study, we reveal the role of oncogenic TF FOXA1 in regulating rRNA transcription within nucleolar organization regions. By analyzing human TFs in prostate cancer clinical datasets and nucleolar proteomics data, we identified that FOXA1 is partially localized in the nucleolus and correlated with global protein translation. Our extensive FOXA1 ChIP-Seq analysis provides robust evidence of FOXA1 binding across rDNA repeats in prostate cancer cell lines, primary tumors, and castration-resistant variants. Notably, FOXA1 occupancy at rDNA repeats correlates with histone modifications associated with active transcription, namely H3K27ac and H3K4me3. Reducing FOXA1 expression results in decreased transactivation at rDNA, subsequently diminishing global protein synthesis.
CONCLUSIONS: Our results suggest FOXA1 regulates aberrant ribosome biogenesis downstream of oncogenic signaling in prostate cancer.

Diaspididae are one of the most serious small herbivorous insects with piercing-sucking mouth parts and are major economic pests as they attack and destroy perennial ornamentals and food crops. Chemical control is the primary management approach for armored scale infestation. However, chemical insecticides do not possess selectivity in action and not always effective enough for the control of armored scale insects. Our previous work showed that green oligonucleotide insecticides (olinscides) are highly effective against armored and soft scale insects. Moreover, olinscides possess affordability, selectivity in action, fast biodegradability, and a low carbon footprint. Insect pest populations undergo microevolution and olinscides should take into account the problem of insecticide resistance. Using sequencing results, it was found that in the mixed populations of insect pests Dynaspidiotus britannicus Newstead and Aonidia lauri Bouche, predominates the population of A. lauri. Individuals of A. lauri comprised for 80% of individuals with the sequence 3\'-ATC-GTT-GGC-AT-5\' in the 28S rRNA site, and 20% of the population comprised D. britannicus individuals with the sequence 3\'-ATC-GTC-GGT-AT-5\'. We created olinscides Diasp80-11 (5\'-ATG-CCA-ACG-AT-3\') and Diasp20-11 (5\'-ATA-CCG-ACG-AT-3\') with perfect complementarity to each of the sequences. Mortality of insects on the 14th day comprised 98.19 ± 3.12% in Diasp80-11 group, 64.66 ± 0.67% in Diasp20-11 group (p < 0.05), and 3.77 ± 0.94% in the control group. Results indicate that for maximum insecticidal effect it is necessary to use an oligonucleotide insecticide that corresponds to the dominant species. Mortality in Diasp80-11 group was accompanied with significant decrease in target 28S rRNA concentration and was 8.44 ± 0.14 and 1.72 ± 0.36 times lower in comparison with control (p < 0.05) on the 10th and 14th days, respectively. We decided to make single nucleotide substitutions in Diasp20-11 olinscide to understand which nucleotide will play the most important role in insecticidal effect. We created three sequences with single nucleotide transversion substitutions at the 5\'-end - Diasp20(5\')-11 (A to T), 3\'-end - Diasp20(3\')-11 (T to A), and in the middle of the sequence - Diasp20(6)-11 (6th nitrogenous base of the sequence; G to C), respectively. As a result, mortality of mixed population of the field experiment decreased and comprised 53.89 ± 7.25% in Diasp20(5\')-11 group, 40.68 ± 4.33% in Diasp20(6)-11 group, 35.74 ± 5.51% in Diasp20(3\')-11 group, and 3.77 ± 0.94% in the control group on the 14th day. Thus, complementarity of the 3\'-end nucleotide to target 28S rRNA was the most important for pronounced insecticidal effect (significance of complementarity of nucleotides for insecticidal effect: 5\' nt < 6 nt < 3\' nt). As was found in our previous research works, the most important rule to obtain maximum insecticidal effect is complete complementarity to the target rRNA sequence and maximum coverage of target sequence in insect pest populations. However, in this article we also show that the complementarity of 3\'-end is a second important factor for insecticidal potential of olinscides. Also in this article we propose 2-step DNA containment mechanism of action of olinscides, recruiting RNase H. The data obtained indicate the selectivity of olinscides and at the same time provide a simple and flexible platform for the creation of effective plant protection products, based on antisense DNA oligonucleotides.

Contamination of the environment due to speedup of anthropogenic activities has become a serious threat to modern humanity. Among the contaminants, the new emerging concern is the heavy metal (HM) contamination in the environment. Because the persistence and harmfulness of heavy metals affect the ecosystem and the health of plants, animals, and humans, they are the most toxic substances in the environment. Among them, Arsenic (As) emerged as major environmental constraint leading to enormous negative effects on the plant, animal, and human health. Even in minute quantity, As is known to cause various critical diseases in humans and toxicity in plants. Research was performed to observe the capability of plant growth-promoting strains of bacteria in enhancing Zea mays (L.) growth in arsenic polluted soil. Total 30 bacterial strains were isolated from the polluted soils, screened for plant growth promotion potential and arsenic tolerance. Eighteen isolates showed resistance to different levels of sodium arsenate (ranging from 0 to 50 mM) in agar plate using LB media. Of 18 isolates, 83.3% produced IAA, methyl red, and hydrogen cyanide; 55.5% exhibited catalase activity; 61.1% showed siderophore production; 88.8% showed phosphate solubilization; and 44.4% showed oxidase, Voges proskauer activity, and KOH solubility. The most efficient isolates SR3, SD5, and MD3 with significant arsenic tolerance and plant growth-promoting (PGP) activity were examined via sequencing of amplified 16S rRNA gene. Isolates of bacteria, i.e., SR3, SD5, and MD3, showing multiple PGP-traits were identified as Bacillus pumilus (NCBI accession number: OR459628), Paenibacillus faecalis (NCBI accession number: OR461560), and Pseudochrobactrum asaccharolyticum (NCBI accession number: OR458922), respectively. Maize seeds treated with these PGPR strains were grown in pots contaminated with 50 ppm and 100 ppm sodium arsenate. Compared to untreated arsenic stressed plants, bacterial inoculation P. asaccharolyticum (MD3) resulted 20.54%, 18.55%, 33.45%, 45.08%, and 48.55% improvement of photosynthetic pigments (carotenoid content, chlorophyll content, stomatal conductance (gs), substomatal CO2, and photosynthetic rate), respectively. Principal component analysis explained that first two components were more than 96% of the variability for each tested parameter. The results indicate that in comparison to other isolates, P. asaccharolyticum isolate can be used as efficient agent for improving maize growth under arsenic polluted soil.

Zygotic genome activation (ZGA) is a critical event in early embryonic development, and thousands of genes are involved in this delicate and sophisticated biological process. To date, however, only a handful of these genes have revealed their core functions in this special process, and therefore the roles of other genes still remain unclear. In the present study, we used previously published transcriptome profiling to identify potential key genes (candidate genes) in minor ZGA and major ZGA in both human and mouse specimens, and further identified the conserved genes across species. Our results showed that 887 and 760 genes, respectively, were thought to be specific to human and mouse in major ZGA, and the other 135 genes were considered to be orthologous genes. Moreover, the conserved genes were most enriched in rRNA processing in the nucleus and cytosol, ribonucleoprotein complex biogenesis, ribonucleoprotein complex assembly and ribosome large subunit biogenesis. The findings of this first comprehensive identification and characterization of candidate genes in minor and major ZGA provide relevant insights for future studies on ZGA.

Ribosomal RNA (rRNA) gives rise to non-random small RNA fragments known as ribosomal-derived small RNAs (rsRNAs), which despite their biological importance, have been relatively understudied in comparison to other short non-coding RNAs. There exists a compelling necessity to develop a methodology for the identification, categorization, and quantification of rsRNAs from small RNA sequencing (sRNA-seq) data sets, considering the unique characteristics of ribosomal RNA (rRNA). To bridge this gap, we introduce \'rsRNAfinder\' a specialized pipeline designed within the Snakemake framework. This analytical approach enables robust identification of rsRNAs using sRNA-seq datasets from Arabidopsis thaliana. Our methodology constitutes an integrated bioinformatic pipeline designed for different kinds of analysis.1.sRNA-seq data analysis: It performs in-depth analysis of reference-aligned sRNA-seq data, facilitating rsRNA annotation and quantification.2.Parametric reporting: Our pipeline provides comprehensive reports encompassing key parameters such as rsRNA size distributions, strandedness, genomic origin, and source rRNA origin.3.Illustrative validation: We have demonstrated the utility of our approach by conducting comprehensive rsRNA annotation in Arabidopsis thaliana. This validation reveals unique rsRNAs originating from all rRNA types, each of them distinguished by distinct identity, abundance, and length.