Internal oligo(dT) priming in bulk and single cell RNA sequencing

Significant advances in RNA sequencing have been recently made possible by the use of oligo(dT) primers for simultaneous mRNA enrichment and reverse transcription priming. The associated increase in efficiency has enabled more economical bulk RNA sequencing methods as well as the advent of high throughput single cell RNA sequencing, now already one of the most widely adopted new methods in the study of transcriptomics. However, the effects of off-target oligo(dT) priming on gene expression quantification have not been fully appreciated. In the present study, we describe the extent, the possible causes, and the consequences of internal oligo(dT) priming across multiple publicly available datasets obtained from a variety of bulk and single cell RNA sequencing platforms. In order to explore and address this issue, we developed a computational algorithm for identification of sequencing read alignments that likely resulted from internal oligo(dT) priming and their subsequent removal from the data. Directly comparing filtered datasets to those obtained by an alternative method reveals significant improvements in gene expression measurement. Finally, we infer a list of genes whose expression quantification is most likely to be affected by internal oligo(dT) priming.

Synapse-specific Lrp4 mRNA enrichment requires Lrp4/MuSK signaling, muscle activity and Wnt non-canonical pathway

Background The neuromuscular junction (NMJ) is a peripheral synapse critical to muscle contraction. Like acetylcholine receptors (AChRs), many essential proteins of NMJ are extremely concentrated at the postjunctional membrane. However, the mechanisms of synapse-specific concentration are not well understood; furthermore, it is unclear whether signaling molecules critical to NMJ formation and maintenance are also locally transcribed. Results We studied the β-gal activity encoded by a lacZ cassette driven by the promoter of the Lrp4 gene. As reported for Lrp4 mRNA, β-gal was in the central region in embryonic muscles and at the NMJ after its formation. However, β-gal was no longer in the central areas of muscle fibers in Lrp4 or MuSK mutant mice, indicating a requirement of Lrp4/MuSK signaling. This phenotype could be rescued by transgenic expression of LRP4 with a transmembrane domain but not soluble ECD in Lrp4 mutant mice. β-gal and AChR clusters were distributed in a broader region in lacZ/ECD than that of heterozygous lacZ/+ mice, indicating an important role of the transmembrane domain in Lrp4 signaling. Synaptic β-gal activity became diffused after denervation or treatment with µ-conotoxin, despite its mRNA was increased, indicating synaptic Lrp4 mRNA enrichment requires muscle activity. β-gal was also diffused in aged mice but became re-concentrated after muscle stimulation. Finally, Lrp4 mRNA was increased in C2C12 myotubes by Wnt ligands in a manner that could be inhibited by RKI-1447, an inhibitor of ROCK in Wnt non-canonical signaling. Injecting RKI-1447 into muscles of adult mice diminished Lrp4 synaptic expression. Conclusions This study demonstrates that synapse-specific enrichment of Lrp4 mRNA requires a coordinated interaction between Lrp4/MuSK signaling, muscle activity, and Wnt non-canonical signaling. Thus, the study provides a new mechanism for Lrp4 mRNA enrichment. It also provides a potential target for the treatment of NMJ aging and other NMJ-related diseases.

PRKCA Overexpression Is Frequent in Young Oral Tongue Squamous Cell Carcinoma Patients and Is Associated with Poor Prognosis

Oral tongue squamous cell carcinomas (OTSCCs) have an increasing incidence in young patients, and many have an aggressive course of disease. The objective of this study was to identify candidate prognostic protein markers associated with early-onset OTSCC. We performed an exploratory screening for differential protein expression in younger (≤45 years) versus older (>45 years) OTSCC patients in The Cancer Genome Atlas (TCGA) cohort (n = 97). Expression of candidate markers was then validated in an independent Austrian OTSCC patient group (n = 34) by immunohistochemistry. Kaplan–Meier survival estimates were computed, and genomic and mRNA enrichment in silico analyses were performed. Overexpression of protein kinase C alpha (PRKCA) was significantly more frequent among young patients of both the TCGA (p = 0.0001) and the Austrian cohort (p = 0.02), associated with a negative anamnesis for alcohol consumption (p = 0.009) and tobacco smoking (p = 0.02) and poorer overall survival (univariate p = 0.02, multivariate p< 0.01). Within the young subgroup, both overall and disease-free survival were significantly decreased in patients with PRKCA overexpression (both p < 0.001). TCGA mRNA enrichment analysis revealed 332 mRNAs with significant differential expression in PRKCA-upregulated versus PRKCA-downregulated OTSCC (all FDR ≤ 0.01). Our findings suggest that PRKCA overexpression may be a hallmark of a novel molecular subtype of early-onset alcohol- and tobacco-negative high-risk OTSCC. Further analysis of the molecular PRKCA interactome may decipher the underlying mechanisms of carcinogenesis and clinicopathological behavior of PRKCA-overexpressing OTSCC.

A putative stem-loop structure in Drosophila crumbs is required for mRNA localisation in epithelia and germline cells

Crumbs (Crb) is an evolutionarily conserved transmembrane protein localised in the apical membrane of epithelial cells. Loss or mis-localisation of Crb is often associated with disruption of apico-basal cell polarity. crb mRNA is also apically enriched in epithelial cells, and, as shown here, accumulates in the oocyte of developing egg chambers. We narrowed down the Localization Element (LE) of crb mRNA to 47 nucleotides forming a putative stem-loop structure, suggesting to be recognised by Egalitarian (Egl). Mutations in conserved nucleotides abrogate apical transport. crb mRNA enrichment in the oocyte is affected in egl mutant egg chambers. A CRISPR based genomic deletion of the crb locus that includes the LE disrupts asymmetric crb mRNA localisation in epithelia and prevents its accumulation in the oocyte during early stages of oogenesis, but does not affect Crb protein localisation in embryonic and follicular epithelia. However, flies lacking the LE show ectopic Crb protein expression in the nurse cells. These data suggest an additional role of the Drosophila 3’-UTR in regulating translation in a tissue specific manner.

Single-molecule analysis of endogenous β-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons

During embryonic nervous system assembly, mRNA localization is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localization patterns across the axon. However, little is known about how mRNAs move in axons or how these patterns are generated. Here, we couple molecular beacon technology with highly inclined and laminated optical sheet microscopy to image single molecules of identified endogenous mRNA in growing axons. By combining quantitative single-molecule imaging with biophysical motion models, we show that β-actin mRNA travels mainly as single copies and exhibits different motion-type frequencies in different axonal subcompartments. We find that β-actin mRNA density is fourfold enriched in the growth cone central domain compared with the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modeling we further demonstrate that directional differences in motor-driven mRNA transport speeds are sufficient to generate β-actin mRNA enrichment at the growth cone. Our results provide insight into how mRNAs are trafficked in axons and a mechanism for generating different mRNA densities across axonal subcompartments.

Metatranscriptome of an Anaerobic Benzene-Degrading, Nitrate-Reducing Enrichment Culture Reveals Involvement of Carboxylation in Benzene Ring Activation

ABSTRACTThe enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the familyPeptococcaceaefrom the orderClostridialeswere essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to anAzoarcusspecies being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to thePeptococcaceae, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degradingPeptococcaceaestrain and a benzoate-degrading denitrifyingAzoarcusstrain for the complete catabolism of benzene with nitrate as the terminal electron acceptor.

Periostin as a Biomarker of the Amniotic Membrane

Tracing the precise developmental origin of amnion and amnion-derived stem cells is still challenging and depends chiefly on analyzing powerful genetic model amniotes like mouse. Profound understanding of the fundamental differences in amnion development in both the disc-shaped primate and human embryo and the cup-shaped mouse embryo is pivotal in particular when sampling amniotic membrane from nonprimate species for isolating candidate amniotic stem cells. The availability of molecular marker genes that are specifically expressed in the amniotic membrane and not in other extraembryonic membranes would be instrumental to validate unequivocally the starting material under investigation. So far such amniotic markers have not been reported. We postulated that bone morphogenetic protein (BMP) target genes are putative amniotic membrane markers mainly because deficiency in one of several components of the BMP signaling cascade in mice has been documented to result in defective development of the early amnion. Comparative gene expression analysis of acknowledged target genes for BMP in different extraembryonic tissues, combined within situhybridization, identifiedPeriostin (Postn)mRNA enrichment in amnion throughout gestation. In addition, we identify and propose a combination of markers as transcriptional signature for the different extraembryonic tissues in mouse.

Two adjacent residues in staphylococcal enterotoxins A and E determine T cell receptor V beta specificity.

The T cell receptor (TCR) V beta-determining region of two bacterial superantigens, staphylococcal enterotoxin A (SEA) and SEE, has been mapped to the COOH-terminal region of SEA and SEE using a panel of recombinant SEA/SEE hybrids. Total TCR V beta mRNA enrichment in human peripheral blood T cell cultures was determined by a novel single-tube amplification technique using a redundant V beta-specific primer. SEA routinely enriched mRNA coding for hV beta 1.1, 5.3, 6.3, 6.4, 6.9, 7.3, 7.4, and 9.1, while SEE, which is 83% homologous to SEA, enriched hV beta 5.1, 6.3, 6.4, 6.9, and 8.1 mRNA. Exchanging residues 206 and 207 was sufficient to convert in toto the TCR V beta response of human peripheral T lymphocytes. In addition, an SEA-reactive murine T cell line, SO3 (mV beta 17), unresponsive to wild-type SEE responded to SEE-S206N207, while an SEE-specific human T cell line, Jurkat (hV beta 8.1), unresponsive to SEA was stimulated strongly by SEA-P206D207. Exchanging all other regions of SEA and SEE except residues 206 and 207 did little to change the V beta response. Thus, the V beta binding region appears to be a stable, discrete domain localized within the COOH-terminal region that is largely unaffected by the considerable amino acid variability between SEA and SEE. This region may interact directly with TCR V beta.