RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5′ and 3′ untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels.antisense RNA | overlapping transcription | RNA processing | posttranscriptional regulation | microRNA F or many years, the catalog of transcripts (transcriptome) produced by bacterial cells was limited to the transcription products of known annotated genes (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). In the past 10 years, the development of new approaches based on high-resolution tiling arrays and RNA deep sequencing (RNA-seq) has uncovered that a significant proportion (depending on the study, varying between 3% and >50%) of protein coding genes are also transcribed from the reverse complementary strand (1-17). In most cases, overlapping transcription generates a noncoding antisense transcript whose size can vary between various tens of nucleotides (cisencoded small RNAs) to thousands of nucleotides (antisense RNAs). The antisense transcript can cover the 5′ end, 3′ end, middle, entire gene, or even various contiguous genes. Alternatively, overlapping transcription can also be due to the overlap between long 5′ or 3′ UTRs of mRNAs transcribed in the opposite direction. Independent of the mechanism by which it is generated, overlapping transcription has been proposed to affect the expression of the target gene at different levels [for review, see Thomason and Storz (18)]. These mechanisms include: (i) the overlapped transcript affects the stability of the target RNA by either promoting (RNA degradation) or blocking (RNA stabilization) cleavage by endoribonucleases or exoribonucleases; (ii) the overlapped transcript induces a change in the structure of the mRNA that affects transcription termination (transcription attenuation); (iii) the overlapped transcript prevents RNA polymerase from binding or extending the transcript encoded in the opposite strand (transcription interference); and (iv) the overl...

BackgroundDemyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage.Methods/Principal FindingsTo monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage.ConclusionThe model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.