rRNA Degradation Research Articles

Quorum sensing regulates rRNA synthesis in Saccharomyces cerevisiae.

Ribosome biogenesis requires the concerted activities of three nuclear RNA polymerases, (Pols) I, II, and III, to produce 25S, 18S and 5.8S ribosomal RNA (rRNA), messenger RNA (mRNA) encoding ribosomal proteins, and the 5S rRNA, respectively. The rRNA is processed and modified before being assembled with ribosomal proteins to produce a ribosome. Ribosome biogenesis requires extensive energetic investment by the cell, so it is critical that this process is tightly regulated in accord with cellular growth potential. Previous work revealed that rRNA synthesis in Saccharomyces cerevisiae is repressed prior to the cells shift from active growth (log phase) to limited/static growth (stationary phase). The mechanism(s) by which cells anticipate imminent stationary phase are unknown. In this study, we demonstrate that growing cells produce one or more compounds that accumulate in the growth medium, and that this compound induces repression of rRNA synthesis. When cells encounter this compound, rRNA synthesis is rapidly repressed. We further show that subunits of Pols I and II are degraded during the transition from log to stationary phase growth, but this degradation does not account for the observed repression of rRNA synthesis. Interestingly, repression of rRNA synthesis by spent media requires the nuclear exosome, implying that spent media stimulates rapid rRNA degradation. Together, these data suggest that yeast use quorum sensing to regulate rRNA synthesis in anticipation of high cell density in stationary phase.

Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese

Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.

Hfq and RNase R Mediate rRNA Processing and Degradation in a Novel RNA Quality Control Process.

RNA quality control pathways are critical for cell survival. Here, we describe a new surveillance process involved in the degradation of highly structured and stable ribosomal RNAs. The results demonstrated that the RNA chaperone Hfq and the 3'-5' exoribonuclease R mediate the elimination of detrimental rRNA fragments and are required for the correct processing of rRNA precursors. Escherichia coli cells lacking both Hfq and RNase R accumulate a high level of 16S- and 23S-derived rRNA fragments. Hfq and RNase R were also shown to participate in the maturation of 16S and 23S rRNA precursors. This correlates with the fact that in the absence of Hfq and RNase R, there are severe ribosome assembly defects and a sharp reduction in 70S ribosome levels. Hfq and RNase R may act independently or in a complex, as protein interaction studies revealed that these RNA-binding proteins can associate. This is the first demonstration that the well-conserved Hfq and RNase R proteins act on common regulatory pathways, unraveling previously unknown mechanisms of rRNA surveillance with important consequences for translation and cell survival.IMPORTANCE Quality control pathways that oversee the quality of stable RNA molecules are critical for the cell. In this work, we demonstrate, for the first time, a functional link between Hfq and RNase R in the processing and degradation of the highly structured rRNAs. These RNA-binding proteins are required for the maturation of 16S and 23S rRNAs and correct ribosome assembly. Furthermore, they participate in the degradation of rRNAs and clearance of toxic rRNA fragments from the cell. Our studies have also shown that Hfq and RNase R can form a complex. In summary, the cooperation between Hfq and RNase R in metabolic pathways of stable RNAs may represent a broader mechanism of RNA quality control, given the high conservation of these RNA-binding proteins throughout evolution.

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.

CDE-1 suppresses the production of risiRNA by coupling polyuridylation and degradation of rRNA

BackgroundModification of RNAs, particularly at the terminals, is critical for various essential cell processes; for example, uridylation is implicated in tumorigenesis, proliferation, stem cell maintenance, and immune defense against viruses and retrotransposons. Ribosomal RNAs can be regulated by antisense ribosomal siRNAs (risiRNAs), which downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3′-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3′ to 5′ exoribonuclease SUSI-1(ceDIS3L2).ResultsHere, we found that CDE-1, one of the three C.elegans polyuridylation polymerases (PUPs), is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates Argonaute-associated 22G-RNAs, 26S, and 5.8S rRNAs at the 3′-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with these results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi.ConclusionsThis work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.

How Tupanvirus Degrades the Ribosomal RNA of Its Amoebal Host? The Ribonuclease T2 Track.

Tupanviruses are giant viruses recently discovered in Brazil from extreme environments: Tupanvirus soda lake (TPV-SL) and Tupanvirus deep ocean (TPV-DO). Unexpected features in Tupanviruses is the cytotoxic effect observed during infection, where the virus degrades the ribosomal RNA (rRNA) of its amoebal host. Interestingly, only TPV-SL causes this rRNA shutdown. We performed a genomic comparison of the two strains to determine potential modifications explaining the absence of rRNA degradation by TPV-DO. Whole genome comparisons were performed as well as more in-depth analysis at the gene level. We also calculated selective pressure on the orthologous genes between the two viruses. Our computational and evolutionary investigations revealed a potential target: a ribonuclease T2. These enzymes are known to be involved in cellular RNA catabolism such as in lysosomal degradation of rRNA. Our results suggest a functional ribonuclease localized in acid compartment closely related to ribonuclease T2 from eukaryotes. Silencing of the RNAse T2 gene of TPV-SL abolished its rRNA shutdown ability thereby correlating in silico assumption to the experimental evidence. In conclusion, all our results pointed to RNAse T2 as a target for explaining the difference for rRNA degradation ability between both strains.

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.

Ccm1p is a 15S rRNA primary transcript processing factor as elucidated by a novel in vivo system in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the mitoribosomal RNA of the minor subunit, 15S rRNA, is transcribed as a bicistronic transcript along with tRNAW. 5' and 3' sequences flanking the mature transcript must be removed by cleavage at the respective junctions before incorporating it into the mitoribosome. An in vivo dose-response triphasic system was created to elucidate the role of Ccm1p in the processing of 15S rRNA: Ccm1p supply ("On"), deprivation ("Off"), and resupply ("Back on"). After 72h under "Off" status, the cells started to exhibit a complete mutant phenotype as assessed by their lack of growth in glycerol medium, while keeping their mitochondrial DNA integrity (ρ+). Full functionality of mitochondria was reacquired upon "Back on." 15S rRNA levels and phenotype followed the Ccm1p intramitochondrial concentrations throughout the "On-Off-Back on" course. Under "Off" status, cells gradually accumulated unprocessed 5' and 3' junctions, which reached significant levels at 72-96h, probably due to a saturation of the mitochondrial degradosome (mtEXO). The Ccm1p/mtEXO mutant (Δccm1/Δdss1) showed a copious accumulation of 15S rRNA primary transcript forms, which were cleaved upon Ccm1p resupply. The gene that codes for the RNA component of RNase P was conserved in wild-type and mutant strains. Our results indicate that Ccm1p is crucial in processing the 15S rRNA primary transcript and does not stabilize the already mature 15S rRNA. Consequently, failure of this function in Δccm1 cells results, as it happens to any other unprocessed primary transcripts, in total degradation of 15S rRNA by mtEXO, whose mechanism of action is discussed.

Short-term kinetics of rRNA degradation in Escherichia coli upon starvation for carbon, amino acid or phosphate.

Ribosomes are absolutely essential for growth but are, moreover, energetically costly to produce. Therefore, it is important to adjust the cellular ribosome levels according to the environmental conditions in order to obtain the highest possible growth rate while avoiding energy wastage on excess ribosome biosynthesis. Here we show, by three different methods, that the ribosomal RNA content of Escherichia coli is downregulated within minutes of the removal of an essential nutrient from the growth medium, or after transcription initiation is inhibited. The kinetics of the ribosomal RNA reduction vary depending on which nutrient the cells are starved for. The number of ribosomes per OD unit of cells is roughly halved after 80min of starvation for isoleucine or phosphate, while the ribosome reduction is less extensive when the cells are starved for glucose. Collectively, the results presented here support the simple model proposed previously, which identifies the inactive ribosomal subunits as the substrates for degradation, since the most substantial rRNA degradation is observed under the starvation conditions that most directly affect the protein synthesis.

Identification of the minimal AcMNPV P143 protein region responsible for triggering apoptosis and rRNA degradation of Bombyx mori cells

Bombyx mori cells induce antiviral responses including global protein synthesis shutdown, rRNA degradation, and apoptosis upon infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We previously demonstrated that five and six amino acid residues located at positions between 514 and 599 of AcMNPV P143 (Ac-P143) protein are important for induction of apoptosis and rRNA degradation, respectively. However, it remains unexplored whether other residues of Ac-P143 protein also participate in antiviral immune responses. Here, we conducted transient expression analysis using a number of Ac-P143 protein deletion and truncation mutants and found that some of the N-terminal 413 residues (amino acids 1–413), besides previously identified residues between amino acids 514 and 599, are indispensable, whereas C-terminal 622 residues (amino acids 600–1221) are dispensable, for Ac-P143 protein to induce apoptosis or rRNA degradation. In addition, we found that the N-terminal 413 sequence (amino acids 1-413) of Ac-P143 protein can be substituted with corresponding BmNPV P143 (Bm-P143) protein sequence. Further analysis demonstrated that mutant Ac-P143 protein consisting of 275 residues (amino acids 325-599), but not 274 residues (amino acids 326-599) lacking glutamine residue at position 325 (Q325), is sufficient for triggering apoptosis and rRNA degradation of B. mori cells. These 275 residues are located outside the region of DNA helicase motifs of Ac-P143 protein, indicating that induction of apoptosis or rRNA degradation occurs independently of viral DNA replication-related function of the Ac-P143 protein. Moreover, Ac-P143(325–599/Q325A) and Ac-P143(1–599/Q325A) proteins harboring Q325A substitution retain the ability to induce apoptosis and rRNA degradation in B. mori cells. These findings suggest that the Ac-P143 protein needs minimal sequence length starting from the Q325 residue that contains a specific effector domain to induce apoptosis and rRNA degradation.

Sequential Ubiquitination of Ribosomal Protein uS3 Triggers the Degradation of Non-functional 18S rRNA

18S non-functional rRNA decay (NRD) eliminates non-functional 18S rRNA with deleterious mutations in the decoding center. Dissociation of the non-functional 80S ribosome into 40S and 60S subunits is aprerequisite step for degradation of the non-functional 18S rRNA. However, the mechanisms by which the non-functional ribosome is recognized and dissociated into subunits remain elusive. Here, we report that the sequential ubiquitination of non-functional ribosomes is crucial for subunit dissociation. 18S NRD requires Mag2-mediated monoubiquitination followed by Hel2- and Rsp5-mediated K63-linked polyubiquitination of uS3 at the 212th lysine residue. Determination of the aberrant 18S rRNA levels in sucrose gradient fractions revealed that the subunit dissociation of stalled ribosomes requires sequential ubiquitination of uS3 by E3 ligases and ATPase activity of Slh1 (Rqt2), as well as Asc1 and Dom34. We propose that sequential uS3 ubiquitination of the non-functional 80S ribosome induces subunit dissociation by Slh1, leading to degradation of the non-functional 18S rRNA.

OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages

Upon viral infection, the 2′, 5′-oligoadenylate synthetase (OAS)-ribonuclease L (RNaseL) system works to cleave viral RNA, thereby blocking viral replication. However, it is unclear whether OAS proteins have a role in regulating gene expression. Here, we show that OAS1 and OAS3 act as negative regulators of the expression of chemokines and interferon-responsive genes in human macrophages. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology was used to engineer human myeloid cell lines in which the OAS1 or OAS3 gene was deleted. Neither OAS1 nor OAS3 was exclusively responsible for the degradation of rRNA in macrophages stimulated with poly(I:C), a synthetic surrogate for viral double-stranded (ds)RNA. An mRNA sequencing analysis revealed that genes related to type I interferon signaling and chemokine activity were increased in OAS1−/− and OAS3−/− macrophages treated with intracellular poly(I:C). Indeed, retinoic-acid-inducible gene (RIG)-I- and interferon-induced helicase C domain-containing protein (IFIH1 or MDA5)-mediated induction of chemokines and interferon-stimulated genes was regulated by OAS3, but Toll-like receptor 3 (TLR3)- and TLR4-mediated induction of those genes was modulated by OAS1 in macrophages. However, stimulation of these cells with type I interferons had no effect on OAS1- or OAS3-mediated chemokine secretion. These data suggest that OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages.

Organelle-associated rRNA Degradation.

Cytosolic rRNAs are highly dynamic and can be degraded under conditions such as apoptosis, starvation and magnesium depletion. The degradation is also related to their specific localization, as fractions of cytosolic ribosomes are localized on the surfaces of intracellular organelles, such as endoplasmic reticulum (ER) and mitochondria. Such localized translation facilitates translocation of nascent proteins into these organelles co-translationally, contributing to fast responses to cellular stresses and precise regulations of the organelle. Here, we describe a protocol to establish the in organello system to investigate rRNA degradation on mitochondrial outer membrane or ER. The protocol consists of organelle isolation, rRNA degradation on organelles and agarose gel electrophoresis to examine the remaining rRNAs.

Degradation of β-Actin mRNA and 18S rRNA in mouse spleen cells after death

We observed degradation of β-actin mRNA and 18S rRNA in mouse spleen cells under constant temperature conditions in the different temperature group during postmortem intervals (PMIs) of 0–72 h. Thirty-nine mice were sacrificed by cervical dislocation and kept at constant temperatures of 10°C, 15°C, 20°C, 25°C, and 30°C. From 0 to 72 h after death, total RNA in spleen cells was extracted every 6 h. The cycle threshold (Ct) values of β-actin mRNA and 18S rRNA were obtained by real-time-quantitative polymerase chain reaction. The results showed that, under the conditions of different and constant temperatures after mouse death at 72 h, the Ct values of β-actin and 18S, Ct ratios of β-actin to 18S, and relative ratios of β-actin to 18S were significantly correlated with PMI. In addition, the relative degradation rates of β-actin and 18S appeared to change from fast to slow with the increase of temperature. By interpolation and fitting analysis of the data, we obtained a ternary quintic equation of the relationship between the change in the relative ratios and PMI, which can be used to infer PMI within a certain temperature range (10°C–30°C).

Antiviral immune responses of Bombyx mori cells during abortive infection with Autographa californica multiple nucleopolyhedrovirus

Lepidopteran cells rely on multiple antiviral responses to defend against baculovirus infections, including apoptosis, global protein synthesis shutdown, and rRNA degradation. Here, we characterized apoptosis and rRNA degradation in Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Bombyx mori cells, a system resulting in abortive infection, in relation to viral DNA replication and viral late gene expression. RNAi-mediated silencing of viral DNA replication-related genes prevented apoptosis, but not rRNA degradation, in B. mori cells infected with p35-deficient AcMNPV. Additionally, AcMNPV, but not B. mori nucleopolyhedrovirus (BmNPV), drastically reduced B. mori cellular iap1 transcript levels and p35-deficient AcMNPV induced more prominent apoptosis than did p35-deficient BmNPV. These results, together with previous results that global protein synthesis shutdown follows viral DNA replication, demonstrate that rRNA degradation is the primary antiviral response that abolishes productive AcMNPV infection of B. mori cells. Our results also demonstrate that B. mori cells induce apoptosis to a different extent depending on NPV species.

Role of ribosome assembly in Escherichia coli ribosomal RNA degradation.

DEAD-Box proteins (DBPs) constitute a prominent class of RNA remodeling factors that play a role in virtually all aspects of RNA metabolism. To better define their cellular functions, deletions in the genes encoding each of the Escherichia coli DBPs were combined with mutations in genes encoding different Ribonucleases (RNases). Significantly, double-deletion strains lacking Ribonuclease R (RNase R) and either the DeaD or SrmB DBP were found to display growth defects and an enhanced accumulation of ribosomal RNA (rRNA) fragments. As RNase R is known to play a key role in removing rRNA degradation products, these observations initially suggested that these two DBPs could be directly involved in the same process. However, additional investigations indicated that DeaD and SrmB-dependent rRNA breakdown is caused by delays in ribosome assembly that increase the exposure of nascent RNAs to endonucleolytic cleavage. Consistent with this notion, mutations in factors known to be important for ribosome assembly also resulted in enhanced rRNA breakdown. Additionally, significant levels of rRNA breakdown products could be visualized in growing cells even in the absence of assembly defects. These findings reveal a hitherto unappreciated mechanism of rRNA degradation under conditions of both normal and abnormal ribosome assembly.

Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae

Stress-induced strand breaks in rRNA have been observed in many organisms, but the mechanisms by which they originate are not well-understood. Here we show that a chemical rather than an enzymatic mechanism initiates rRNA cleavages during oxidative stress in yeast (Saccharomyces cerevisiae). We used cells lacking the mitochondrial glutaredoxin Grx5 to demonstrate that oxidant-induced cleavage formation in 25S rRNA correlates with intracellular iron levels. Sequestering free iron by chemical or genetic means decreased the extent of rRNA degradation and relieved the hypersensitivity of grx5Δ cells to the oxidants. Importantly, subjecting purified ribosomes to an in vitro iron/ascorbate reaction precisely recapitulated the 25S rRNA cleavage pattern observed in cells, indicating that redox activity of the ribosome-bound iron is responsible for the strand breaks in the rRNA. In summary, our findings provide evidence that oxidative stress-associated rRNA cleavages can occur through rRNA strand scission by redox-active, ribosome-bound iron that potentially promotes Fenton reaction-induced hydroxyl radical production, implicating intracellular iron as a key determinant of the effects of oxidative stress on ribosomes. We propose that iron binding to specific ribosome elements primes rRNA for cleavages that may play a role in redox-sensitive tuning of the ribosome function in stressed cells.

Role of the novel endoribonuclease SLFN14 and its disease-causing mutations in ribosomal degradation.

Platelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by a low platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants colocalize with ribosomes and mediate rRNA endonucleolytic degradation. Compared to SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients’ platelets. Overall, we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

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.

Apple S-RNase triggers inhibition of tRNA aminoacylation by interacting with a soluble inorganic pyrophosphatase in growing self-pollen tubes invitro.

Apple exhibits S-RNase-based self-incompatibility (SI), in which S-RNase plays a central role in rejecting self-pollen. It has been proposed that the arrest of pollen growth in SI of Solanaceae plants is a consequence of the degradation of pollen rRNA by S-RNase; however, the underlying mechanism in Rosaceae is still unclear. Here, we used S2 -RNase as a bait to screen an apple pollen cDNA library and characterized an apple soluble inorganic pyrophosphatase (MdPPa) that physically interacted with S-RNases. When treated with self S-RNases, apple pollen tubes showed a marked growth inhibition, as well as a decrease in endogenous soluble pyrophosphatase activity and elevated levels of inorganic pyrophosphate (PPi). In addition, S-RNase was found to bind to two variable regions of MdPPa, resulting in a noncompetitive inhibition of its activity. Silencing of MdPPa expression led to a reduction in pollen tube growth. Interestingly, tRNA aminoacylation was inhibited in self S-RNase-treated or MdPPa-silenced pollen tubes, resulting in the accumulation of uncharged tRNA. Furthermore, we provide evidence showing that this disturbance of tRNA aminoacylation is independent of RNase activity. We propose an alternative mechanism differing from RNA degradation to explain the cytotoxicity of the S-RNase apple SI process.