Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin

Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete thus permitting the re-initiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.

TOR mediates the autophagy response to altered nucleotide homeostasis in an RNase mutant

The Arabidopsis thaliana T2 family endoribonuclease RNS2 localizes to the vacuole and functions in rRNA degradation. Loss of RNS2 activity impairs rRNA turnover and leads to constitutive autophagy, a process for degradation of cellular components. Autophagy is normally activated during environmental stress and is important for stress tolerance and homeostasis. Here we show that restoration of cytosolic purine nucleotide levels rescues the constitutive autophagy phenotype of rns2-2 seedlings, whereas inhibition of purine synthesis induces autophagy in wild-type seedlings. rns2-2 seedlings have reduced activity of the target of rapamycin (TOR) kinase complex, a negative regulator of autophagy, and this phenotype is rescued by addition of inosine to increase purine levels. Activation of TOR in rns2-2 by exogenous auxin blocks the enhanced autophagy, indicating a possible involvement of the TOR signaling pathway in the activation of autophagy in the rns2-2 mutant. Our data suggest a model in which loss of rRNA degradation in rns2-2 leads to a reduction in cytoplasmic nucleotide concentrations, which in turn inhibits TOR activity, leading to activation of autophagy to restore homeostasis.

Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation

Ribosome biogenesis is among the founding processes in the cell. During the first stages of ribosome biogenesis, polycistronic precursor of ribosomal RNA passes complex multistage maturation after transcription. Quality control of preribosomal RNA (pre-rRNA) processing is precisely regulated by non-ribosomal proteins and structural features of pre-rRNA molecules, including modified nucleotides. However, many participants of rRNA maturation are still unknown or poorly characterized. We report that RNA m6A methyltransferase Mettl3 interacts with the 5′ external transcribed spacer (5′ETS) of the 47S rRNA precursor and modifies adenosine 196. We demonstrated that Mettl3 knockdown results in the increase of pre-rRNA processing rates, while intracellular amounts of rRNA processing machinery components (U3, U8, U13, U14, and U17 small nucleolar RNA (snoRNA)and fibrillarin, nucleolin, Xrn2, and rrp9 proteins), rRNA degradation rates, and total amount of mature rRNA in the cell stay unchanged. Increased efficacy of pre-rRNA cleavage at A’ and A0 positions led to the decrease of 47S and 45S pre-rRNAs in the cell and increase of mature rRNA amount in the cytoplasm. The newly identified conserved motif DRACH sequence modified by Mettl3 in the 5′-ETS region is found and conserved only in primates, which may suggest participation of m6A196 in quality control of pre-rRNA processing at initial stages demanded by increased complexity of ribosome biogenesis.

Identifying small RNAs derived from maternal- and somatic-type rRNAs in zebrafish development

rRNAs are non-coding RNAs present in all prokaryotes and eukaryotes. In eukaryotes there are four rRNAs: 18S, 5.8S, 28S, originating from a common precursor (45S), and 5S. We have recently discovered the existence of two distinct developmental types of rRNA: a maternal-type, present in eggs and a somatic-type, expressed in adult tissues. Lately, next-generation sequencing has allowed the discovery of new small-RNAs deriving from longer non-coding RNAs, including small-RNAs from rRNAs (srRNAs). Here, we systemically investigated srRNAs of maternal- or somatic-type 18S, 5.8S, 28S, with small-RNAseq from many zebrafish developmental stages. We identified new srRNAs for each rRNA. For 5.8S, we found srRNA consisting of the 5′ or 3′ halves, with only the latter having different sequence for the maternal- and somatic-types. For 18S, we discovered 21 nt srRNA from the 5′ end of the 18S rRNA with a striking resemblance to microRNAs; as it is likely processed from a stem-loop precursor and present in human and mouse Argonaute-complexed small-RNA. For 28S, an abundant 80 nt srRNA from the 3′ end of the 28S rRNA was found. The expression levels during embryogenesis of these srRNA indicate they are not generated from rRNA degradation and might have a role in the zebrafish development.

Nuclear transcriptomes at high resolution using retooled INTACT

Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here we describe transfer of the Isolation of Nuclei from TAgged specific Cell Types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear envelope-targeting domain of the Nuclear Tagging Fusion (NTF) protein with an outer nuclear envelope-anchored domain. This modified NTF was combined with codon optimized E. coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping and profiling of the complete nuclear transcriptome, including a rRNA degradation procedure that minimizes pre-rRNA transcripts. A high-resolution comparison of nuclear and steady-state poly (A)+ transcript populations of seedling root tips confirmed the capture of pre-mRNA and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell-types of rice and other species.Summary:Improved technology and methodology for affinity purification of nuclei and analysis of nuclear transcriptomes, chromatin and other nuclear components.

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

ABSTRACTrRNAs of dormant spores ofBacillus subtiliswere >95% degraded during extended incubation at 50°C, as reported previously (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139–114, 2012, doi:http://dx.doi.org/10.1016/j.cell.2011.11.059), and this was also true of spores ofBacillus megaterium. Incubation of spores of these two species for ∼20 h at 75 to 80°C also resulted in the degradation of all or the great majority of the 23S and 16S rRNAs, although this rRNA degradation was slower than nonenzymatic hydrolysis of purified rRNAs at these temperatures. This rRNA degradation at high temperature generated almost exclusively oligonucleotides with minimal levels of mononucleotides. RNase Y, suggested to be involved in rRNA hydrolysis duringB. subtilisspore incubation at 50°C, did not play a role inB. subtilisspore rRNA breakdown at 80°C. Twenty hours of incubation ofBacillusspores at 70°C also decreased the already minimal levels of ATP in dormant spores 10- to 30-fold, to ≤0.01% of the total free adenine nucleotide levels. Spores depleted of rRNA were viable and germinated relatively normally, often even faster than starting spores. Their return to vegetative growth was also similar to that of untreated spores forB. megateriumspores and slower for heat-treatedB. subtilisspores; accumulation of rRNA took place only after completion of spore germination. These findings thus strongly suggest that protein synthesis is not essential forBacillusspore germination.IMPORTANCEA recent report (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:3486–3495, 2015, doi:http://dx.doi.org/10.1016/j.molcel.2014.12.019) suggested that protein synthesis is essential for early steps in the germination of dormant spores ofBacillus subtilis. If true, this would be a paradigm shift in our understanding of spore germination. We now show that essentially all of the rRNA can be eliminated from spores ofBacillus megateriumorB. subtilis, and these rRNA-depleted spores are viable and germinate as well as or better than spores with normal rRNA levels. Thus, protein synthesis is not required in the process of spore germination.

Antagonism of RNase L Is Required for Murine Coronavirus Replication in Kupffer Cells and Liver Sinusoidal Endothelial Cells but Not in Hepatocytes

ABSTRACTMouse hepatitis virus strain A59 infection of mice is a useful tool for studying virus-host interaction during hepatitis development. The NS2H126Rmutant is attenuated in liver replication due to loss of phosphodiesterase activity, which the wild-type (WT) virus uses to block the 2′,5′-oligoadenylate synthetase (OAS)-RNase L (RNase L) antiviral pathway. The activation of RNase L by NS2H126Ris cell type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We tested the hypothesis that the resident liver macrophages, Kupffer cells (KC), represent the cell type most likely to restrict NS2H126Rand prevent hepatitis. As found previously, A59 and NS2H126Rreplicate similarly in hepatocytes and neither activates RNase L, as assessed by an rRNA degradation assay. In contrast, in KC, A59 exhibited a 100-fold-higher titer than NS2H126Rand NS2H126Rinduced rRNA degradation. Interestingly, in liver sinusoidal endothelial cells (LSEC), the cells that form a barrier between blood and liver parenchymal cells, NS2H126Ractivates RNase L, which limits viral replication. Similar growth kinetics were observed for the two viruses in KC and LSEC from RNase L−/−mice, demonstrating that both use RNase L to limit NS2H126Rreplication. Depletion of KC by gadolinium(III) chloride or of LSEC by cyclophosphamide partially restores liver replication of NS2H126R, leading to hepatitis. Thus, during mouse hepatitis virus (MHV) infection, hepatitis, which damages the parenchyma, is prevented by RNase L activity in both KC and LSEC but not in hepatocytes. This may be explained by the undetectable levels of RNase L as well as by the OASs expressed in hepatocytes.IMPORTANCEMouse hepatitis virus infection of mice provides a useful tool for studying virus-host interactions during hepatitis development. The NS2H126Rmutant is attenuated in liver replication due to loss of phosphodiesterase activity, by which the wild-type virus blocks the potent OAS-RNase L antiviral pathway. RNase L activation by NS2H126Ris cell type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We showed that the hepatocytes that comprise the liver parenchyma do not activate RNase L when infected with NS2H126Ror restrict replication. However, both Kupffer cells (KC) (i.e., the liver-resident macrophages) and the liver sinusoidal endothelial cells (LSEC) which line the sinusoids activate RNase L in response to NS2H126R. These data suggest that KC and LSEC prevent viral spread into the parenchyma, preventing hepatitis. Furthermore, hepatocytes express undetectable levels of OASs and RNase L, which likely explains the lack of RNase L activation during NS2H126Rinfection.