computational-analysis-tutorial-for-chimeric-small-noncoding-rna

Computational Analysis Tutorial for Chimeric Small Noncoding RNA: Target RNA Sequencing Libraries

An understanding of the in vivo gene regulatory interactions of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs), with their target RNAs has ...

Johns Hopkins University School of Medicine

Single-molecule fluorescence  hybridization (smFISH) allows spatial mapping of gene expression. This protocol presents advances in smFISH fidelity and flexibility in intact murine sensory nervous system tissue. An approach using RNAscope probes allows multiplexing, enhanced target specificity, and immunohistochemistry compatibility. Computational strategies increase quantification accuracy of mRNA puncta with a point spread function for clustered transcripts in the dorsal root ganglion and 3D masking for intermingled sciatic nerve cell types. Approaches are validated for mRNAs of modest (Lin28a) and medium (Ppib) steady-state abundance in neurons.

Combined single-molecule fluorescence  hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve.

MicroRNAs (miRNAs) are small non-coding RNAs (sncRNAs) that function in post-transcriptional gene regulation through imperfect base pairing with mRNA targets which results in inhibition of translation and typically destabilization of bound transcripts. Sequence-based algorithms historically used to predict miRNA targets face inherent challenges in reliably reflecting in vivo interactions. Recent strategies have directly profiled miRNA-target interactions by crosslinking and ligation of sncRNAs to their targets within the RNA-induced silencing complex (RISC), followed by high throughput sequencing of the chimeric sncRNA:target RNAs. Despite the strength of these direct profiling approaches, standardized pipelines for effectively analyzing the resulting chimeric sncRNA:target RNA sequencing data are not readily available. Here we present SCRAP, a robust Small Chimeric RNA Analysis Pipeline for the bioinformatic processing of chimeric sncRNA:target RNA sequencing data. SCRAP consists of two parts, each of which are specifically optimized for the distinctive characteristics of chimeric small RNA sequencing reads: first, read processing and alignment and second, peak calling and annotation. We apply SCRAP to benchmark chimeric sncRNA:target RNA sequencing datasets generated by distinct molecular approaches, and compare SCRAP to existing chimeric RNA analysis pipelines. SCRAP has minimal hardware requirements, is cross-platform, and contains extensive annotation to broaden accessibility for processing small chimeric RNA sequencing data and enable insights about the targets of small non-coding RNAs in regulating diverse biological systems.

SCRAP: a bioinformatic pipeline for the analysis of small chimeric RNA-seq data.

Neuropathic pain is a chronic condition arising from damage to somatosensory pathways that results in pathological hypersensitivity. Persistent pain can be viewed as a consequence of maladaptive plasticity which, like most enduring forms of cellular plasticity, requires altered expression of specific gene programs. Control of gene expression at the level of protein synthesis is broadly utilized to directly modulate changes in activity and responsiveness in nociceptive pathways and provides an effective mechanism for compartmentalized regulation of the proteome in peripheral nerves through local translation. Levels of noncoding RNAs (ncRNAs) are commonly impacted by peripheral nerve injury leading to persistent pain. NcRNAs exert spatiotemporal regulation of local proteomes and affect signaling cascades supporting altered sensory responses that contribute to hyperalgesia. This review discusses ncRNAs found in the peripheral nervous system (PNS) that are dysregulated following nerve injury and the current understanding of their roles in pathophysiological pain-related responses including neuroimmune interactions, neuronal survival and axon regeneration, Schwann cell dedifferentiation and proliferation, intercellular communication, and the generation of ectopic action potentials in primary afferents. We review progress in the field beyond cataloging, with a focus on the relevant target transcripts and mechanisms underlying pain modulation by ncRNAs.

Article	Year
Combined single-molecule fluorescence hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve. STAR protocols\| PubMed ID: 34142098	2021
SCRAP: a bioinformatic pipeline for the analysis of small chimeric RNA-seq data. RNA (New York, N.Y.)\| PubMed ID: 36316086	2022
Regulation by noncoding RNAs of local translation, injury responses, and pain in the peripheral nervous system. Neurobiology of pain (Cambridge, Mass.)\| PubMed ID: 36798094	2023

Sreenivas Eadara

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