Salmonella produce microRNA-like RNA fragment Sal-1 in the infected cells to facilitate intracellular survival.

Salmonella have developed a sophisticated machinery to evade immune clearance and promote survival in the infected cells. Previous studies were mostly focused on either bacteria itself or host cells, the interaction mechanism of host-pathogen awaits further exploration. In the present study, we show that Salmonella can exploit mammalian cell non-classical microRNA processing machinery to further process bacterial small non-coding RNAs into microRNA-like fragments. Sal-1, one such fragment with the highest copy number in the infected cells, is derived from Salmonella 5-leader of the ribosomal RNA transcript and has a stem structure-containing precursor. Processing of Sal-1 precursors to mature Sal-1 is dependent on host cell Argonaute 2 (AGO2) but not Dicer. Functionally, depleting cellular Sal-1 strongly renders the Salmonella bacteria less resistant to the host defenses both in vitro and in vivo. In conclusion, we demonstrate a novel strategy for Salmonella evading the host immune clearance, in which Salmonella produce microRNA-like functional RNA fragments to establish a microenvironment facilitating bacterial survival

Limited neuropeptide Y precursor processing in unfavourable metastatic neuroblastoma tumours

Neuropeptide Y (NPY) is found at high concentrations in neural crest-derived tumours and has been implicated as a regulatory peptide in tumour growth and differentiation. Neuroblastomas, ganglioneuromas and phaeochromocytomas with significant concentrations of NPY-like immunoreactivity were investigated for different molecular forms of NPY and for significance of proNPY processing. Gel-permeation chromatography identified intact NPY (1–36) in all tumours, whereas proNPY (69 amino acids) was detected only in control adrenal tissue and malignant neuroblastomas. Purification of NPY-like immunoreactivity in tumour extracts and structural characterization revealed that both NPY (1–36) and the truncated form NPY (3–36) was present. The degree of processing of proNPY to NPY in tumour tissue was lower in advanced neuroblastomas with regional or metastatic spread (stage 3 and 4) (n = 6), (41%, 12–100%, median, range), compared to the less aggressive stage 1, 2 and 4S tumours (n = 12), (93%; 69–100%), (P = 0.012). ProNPY processing of less than 50% was correlated with poor clinical outcome (P = 0.004). MYCN oncogene amplification was also correlated to a low degree of proNPY processing (P = 0.025). In summary, a low degree of proNPY processing was correlated to clinical advanced stage and poor outcome in neuroblastomas. ProNPY/NPY processing generated molecular forms of NPY with known differences in NPY-receptor selectivity, implicating a potential for in vivo modulation of NPY-like effects in tumour tissue. © 2000 Cancer Research Campaig

Identification of novel maize miRNAs by measuring the precision of precursor processing

<p>Abstract</p> <p>Background</p> <p>miRNAs are known to play important regulatory roles throughout plant development. Until recently, nearly all the miRNAs in maize were identified by comparative analysis to miRNAs sequences of other plant species, such as rice and <it>Arabidopsis</it>.</p> <p>Results</p> <p>To find new miRNA in this important crop, small RNAs from mixed tissues were sequenced, resulting in over 15 million unique sequences. Our sequencing effort validated 23 of the 28 known maize miRNA families, including 49 unique miRNAs. Using a newly established criterion, based on the precision of miRNA processing from precursors, we identified 66 novel miRNAs in maize. These miRNAs can be grouped into 58 families, 54 of which have not been identified in any other species. Five new miRNAs were validated by northern blot. Moreover, we found targets for 23 of the 66 new miRNAs. The targets of two of these newly identified miRNAs were confirmed by 5'RACE.</p> <p>Conclusion</p> <p>We have implemented a novel method of identifying miRNA by measuring the precision of miRNA processing from precursors. Using this method, 66 novel miRNAs and 50 potential miRNAs have been identified in maize.</p

The Deafness-Associated Mitochondrial DNA Mutation at Position 7445, Which Affects tRNASer(UCN) Precursor Processing, Has Long-Range Effects on NADH Dehydrogenase Subunit ND6 Gene Expression

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of ~70% in the tRNASer(UCN) level and a decrease of ~45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to ~40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located ~7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells

Characterization of β-amyloid peptide precursor processing by the yeast Yap3 and Mkc7 proteases

AbstractTwo proteases, denoted β- and γ-secretase, process the β-amyloid peptide precursor (APP) to yield the Aβ peptides involved in Alzheimer's disease. A third protein, α-secretase, cleaves APP near the middle of the Aβ sequence and thus prevents Aβ formation. These enzymes have defied identification. Because of its similarity to the systems of mammalian cells the yeast secretory system has provided important clues for finding mammalian processing enzymes. When expressed in Saccharomyces cerevisiae APP is processed by enzymes that possess the specificity of the α-secretases of multicellular organisms. APP processing by α-secretases occurred in sec1 and sec7 mutants, in which transport to the cell surface or to the vacuole is blocked, but not in sec17 or sec18 mutants, in which transport from the endoplasmic reticulum to the Golgi is blocked. Neutralization of the vacuole by NH4Cl did not block α-secretase action. The time course of processing of a pro-α-factor leader-APP chimera showed that processing by Kex2 protease, a Golgi protease that removes the leader, preceded processing by α-secretase. Deletions of the genes encoding the GPI-linked aspartyl proteases Yap3 and Mkc7 decreased α-secretase activity by 56 and 29%, respectively; whereas, the double deletion decreased the activity by 86%. An altered form of APP-695, in which glutamine replaced Lys-612 at the cleavage site, is cleaved by Yap3 at 5% the rate of the wild-type APP. Mkc7 protease cleaved APP (K612Q) at about 20% the rate of wild-type APP. The simplest interpretation of these results is that Yap3 and Mkc7 proteases are α-secretases which act on APP in the late Golgi. They suggest that GPI-linked aspartyl proteases should be investigated as candidate secretases in mammalian tissues

Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide precursor processing carboxypeptidase.

The Saccharomyces cerevisiae KEX1 gene encodes a protease with carboxypeptidase B-like activity involved in K1 and K2 killer toxins and alpha-factor (mating pheromone) precursors processing. The gene has been expressed using the baculovirus/insect cell system, and the KEX1 encoded protein (Kex1p) was purified to apparent homogeneity from detergent-solubilized membrane preparations of insect cells infected with the recombinant virus. The specific activity of the enzyme was enriched 126-fold as compared with the cell lysate, with a recovery of 29%. The NH2-terminal sequence of the purified active enzyme was identical to the predicted sequence after the removal of the signal peptide. This provides evidence that Kex1p, at least in insect cells, is not made as a proenzyme. The optimum pH for activity was 6.0, and the apparent pI value of the protein was below pH 3.0. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides: benzoyl-Phe-Ala-Arg (Km = 284 microM), furylacryloyl (fa)-Ala-Arg (Km = 516 microM), and fa-Ala-Lys (Km = 962 microM). The kinetic data obtained reveals that Kex1p preferentially cleaves the COOH-terminal arginine of peptides over the COOH-terminal lysine. Insect-derived Kex1p processes alpha-factor-Lys-Arg, its known natural substrate, to mature active alpha-factor, and this maturation event takes place in a sequential manner. Furthermore, the enzyme expresses very high affinity for the 15-amino acid-long peptide, alpha-factor-Lys-Arg (Ki = 22 microM), and somewhat lower affinity for the heptapeptides [Leu]enkephalin-Arg-Arg,-Arg-Lys, and [Met]enkephalin-Lys-Lys (Ki = 45, 57, and 81 microM, respectively). The data demonstrate that processing at the COOH terminus of the peptides tested stops after the cleavage of the Arg and/or Lys residues. The specificity of the enzyme for COOH-terminal basic amino acid residues of the peptides used in this study and its high affinity for alpha-factor-Lys-Arg confirms the role that Kex1p plays in polypeptide precursor processing in yeast

Localization of Kex2-like processing endoproteases, furin and PC4, within mouse testis by in situ hybridization

AbstractBy in situ hybridization analysis, we show here the localization of furin and PC4, which are both members of a growing family of endoproteases structurally related to the yeast precursor processing protease Kex2, within mouse testis. Furin transcript was detected in both germ and somatic cells, while PC4 transcript was found only in round spermatids. Proenkephalin transcript was also localized in round spermatids. These observations suggest that, within testis, PC4 is involved in processing of peptide precursors such as proenkephalin and may play a role in regulation of sperm maturation. while furin may serve as a more general processing endoprotease

Precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences

Many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. This processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. In this report we employ an in vitro system to investigate the role of processing in protein import. Intact Neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-phenanthroline. Under these conditions, the processing of the precursors of the beta-subunit of F1-ATPase (F1 beta) and subunit 9 of the F0F1-ATPase was strongly inhibited. Protease- mapping studies indicated that import of the precursor proteins into the mitochondria continued in the absence of processing. Upon readdition of divalent metal to the treated mitochondria, the imported precursors were quantitatively converted to their mature forms. This processing of imported precursors occurred in the absence of a mitochondrial membrane potential and was extremely rapid even at 0 degrees C. This suggests that all or part of the polypeptide chain of the imported precursors had been translocated into the matrix location of the processing enzyme. Localization experiments suggested that the precursor to F1 beta is peripherally associated with the mitochondrial membrane while the precursor to subunit 9 appeared to be tightly bound to the membrane. We conclude that proteolytic processing is not necessary for the translocation of precursor proteins across mitochondrial membranes, but rather occurs subsequent to this event. On the basis of these and other results, a hypothetical pathway for the import of F1 beta and subunit 9 is proposed