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. UtilizingBacillus subtilisas 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.

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.

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.

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.

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