Human RNase P exhibits and controls distinct ribonucleolytic activities required for ordered maturation of tRNA.

Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.

A mutation in POLR3E impairs antiviral immune response and RNA polymerase III.

RNA polymerase (Pol) III has a noncanonical role of viral DNA sensing in the innate immune system. This polymerase transcribes viral genomes to produce RNAs that lead to induction of type I interferons (IFNs). However, the genetic and functional links of Pol III to innate immunity in humans remain largely unknown. Here, we describe a rare homozygous mutation (D40H) in the POLR3E gene, coding for a protein subunit of Pol III, in a child with recurrent and systemic viral infections and Langerhans cell histiocytosis. Fibroblasts derived from the patient exhibit impaired induction of type I IFN and increased susceptibility to human cytomegalovirus (HCMV) infection. Cultured cell lines infected with HCMV show induction of POLR3E expression. However, induction is not restricted to DNA virus, as sindbis virus, an RNA virus, enhances the expression of this protein. Likewise, foreign nonviral DNA elevates the steady-state level of POLR3E and elicits promoter-dependent and -independent transcription by Pol III. Remarkably, the molecular mechanism underlying the D40H mutation of POLR3E involves the assembly of defective initiation complexes of Pol III. Our study links mutated POLR3E and Pol III to an innate immune deficiency state in humans.

Arginine dephosphorylation propels spore germination in bacteria.

Bacterial spores can remain dormant for years but possess the remarkable ability to germinate, within minutes, once nutrients become available. However, it still remains elusive how such instant awakening of cellular machineries is achieved. Utilizing Bacillus subtilis as a model, we show that YwlE arginine (Arg) phosphatase is crucial for spore germination. Accordingly, the absence of the Arg kinase McsB accelerated the process. Arg phosphoproteome of dormant spores uncovered a unique set of Arg-phosphorylated proteins involved in key biological functions, including translation and transcription. Consequently, we demonstrate that during germination, YwlE dephosphorylates an Arg site on the ribosome-associated chaperone Tig, enabling its association with the ribosome to reestablish translation. Moreover, we show that Arg dephosphorylation of the housekeeping σ factor A (SigA), mediated by YwlE, facilitates germination by activating the transcriptional machinery. Subsequently, we reveal that transcription is reinitiated at the onset of germination and its recommencement precedes that of translation. Thus, Arg dephosphorylation elicits the most critical stages of spore molecular resumption, placing this unusual post-translational modification as a major regulator of a developmental process in bacteria.

Characterization and heme oxygenase-1 content of extracellular vesicles in human biofluids.

Heme oxygenase-1 (HO-1), a highly inducible stress protein that degrades heme to biliverdin, carbon monoxide, and free ferrous iron, is increased in blood and other biofluids of subjects with various systemic and neurological disorders. HO-1 does not contain an N-terminal signal peptide and the mechanism responsible for its secretion remains unknown. Extracellular vesicles (EVs) are membrane-bound inclusions that transport microRNAs, messenger RNAs, lipids, and proteins among diverse cellular and extracellular compartments. The objective of the current study was to determine whether EVs in human biofluids contain HO-1, and whether the latter may be transported in EVs from brain to periphery. Total, L1 cell adhesion molecule protein (L1CAM)-enriched (neuron-derived), and glutamate aspartate transporter 1 (GLAST)-enriched (astrocyte-derived) EVs were purified from five different human biofluids (saliva [n = 40], plasma [n = 14], serum [n = 10], urine [n = 10], and cerebrospinal fluid [n = 11]) using polymer precipitation and immuno-affinity-based capture methods. L1CAM-enriched, GLAST-enriched, and L1CAM/GLAST-depleted (LGD) EV, along with EV-depleted (EVD), fractions were validated by nanoparticle tracking analysis, enzyme-linked immunosorbent assay (ELISA), and western blot. HO-1 was assayed in all fractions using ELISA and western blot. The majority of HO-1 protein was localized to LGD, L1CAM-enriched, and GLAST-enriched EVs of all human biofluids surveyed after adjusting for age and sex, with little HO-1 protein detected in EVD fractions. HO-1 protein in human biofluids is predominantly localized to EV compartments. A substantial proportion of EV HO-1 in peripheral human biofluids is derived from the central nervous system and may contribute to the systemic manifestations of various neurological conditions.

Targeted inhibition of WRN helicase, replication stress and cancer.

WRN helicase has several roles in genome maintenance, such as replication, base excision repair, recombination, DNA damage response and transcription. These processes are often found upregulated in human cancers, many of which display increased levels of WRN. Therefore, directed inhibition of this RecQ helicase could be beneficial to selective cancer therapy. Inhibition of WRN is feasible by the use of small-molecule inhibitors or application of RNA interference and EGS/RNase P targeting systems. Remarkably, helicase depletion leads to a severe reduction in cell viability due to mitotic catastrophe, which is triggered by replication stress induced by DNA repair failure and fork progression arrest. Moreover, we present new evidence that WRN depletion results in early changes of RNA polymerase III and RNase P activities, thereby implicating chromatin-associated tRNA enzymes in WRN-related stress response. Combined with the recently discovered roles of RecQ helicases in cancer, current data support the targeting prospect of these genome guardians, as a means of developing clinical phases aimed at diminishing adaptive resistance to present targeted therapies.

Targeted inhibition of WRN helicase by external guide sequence and RNase P RNA.

Human WRN, a RecQ helicase encoded by the Werner syndrome gene, is implicated in genome maintenance, including replication, recombination, excision repair and DNA damage response. These genetic processes and expression of WRN are concomitantly upregulated in many types of cancers. Therefore, targeted destruction of this helicase could be useful for elimination of cancer cells. Here, we provide a proof of concept for applying the external guide sequence (EGS) approach in directing an RNase P RNA to efficiently cleave the WRN mRNA in cultured human cell lines, thus abolishing translation and activity of this distinctive 3'-5' DNA helicase-nuclease. Remarkably, EGS-directed knockdown of WRN leads to severe inhibition of cell viability. Hence, further assessment of this targeting system could be beneficial for selective cancer therapies, particularly in the light of the recent improvements introduced into EGSs.

Human RNase P ribonucleoprotein is required for formation of initiation complexes of RNA polymerase III.

Human RNase P is implicated in transcription of small non-coding RNA genes by RNA polymerase III (Pol III), but the precise role of this ribonucleoprotein therein remains unknown. We here show that targeted destruction of HeLa nuclear RNase P inhibits transcription of 5S rRNA genes in whole cell extracts, if this precedes the stage of initiation complex formation. Biochemical purification analyses further reveal that this ribonucleoprotein is recruited to 5S rRNA genes as a part of proficient initiation complexes and the activity persists at reinitiation. Knockdown of RNase P abolishes the assembly of initiation complexes by preventing the formation of the initiation sub-complex of Pol III. Our results demonstrate that the structural intactness, but not the endoribonucleolytic activity per se, of RNase P is critical for the function of Pol III in cells and in extracts.