Self-incompatibility plays a vital role in angiosperms, by preventing inbreeding depression and maintaining genetic diversity within populations. Following polyploidization, many angiosperm species transition from self-incompatibility to self-compatibility. Here, we investigated the S-locus in Brassicaceae and identified distinct origins for the sRNA loci, SMI and SMI2 (SCR Methylation Inducer 1 and 2), within the S-locus. The SMI loci were found to be widespread in Cruciferae, whereas the SMI2 loci were exclusive to Brassica species. Additionally, we discovered four major S-haplotypes (BnS-1, BnS-6, BnS-7, and BnS-1300) in rapeseed. Overexpression of BnSMI-1 in self-incompatible Brassica napus (\'S-70S1300S6 \') resulted in a significant increase in DNA methylation in the promoter regions of BnSCR-6 and BnSCR-1300, leading to self-compatibility. Conversely, by overexpressing a point mutation of BnSmi-1 in the \'S-70S1300S6 \' line, we observed lower levels of DNA methylation in BnSCR-6 and BnSCR-1300 promoters. Furthermore, the overexpression of BnSMI2-1300 in the \'SI-326S7S6 \' line inhibited the expression of BnSCR-7 through transcriptional repression of the Smi2 sRNA from the BnS-1300 haplotype. Our study demonstrates that the self-compatibility of rapeseed is determined by S-locus sRNA-mediated silencing of SCR after polyploidization, which helps to further breed self-incompatible or self-compatible rapeseed lines, thereby facilitating the utilization of heterosis.

Testicular germ cell tumours (TGCTs) are the most common tumours in young adults of European ancestry. The high heritability and the constantly increased incidence, which has doubled over the last 20 years, strongly suggest that both genetic and environmental factors are likely to shape the TGCT susceptibility. While genome-wide association studies have identified loci associated with TGCT susceptibility, the role played by environmental molecular vectors in TGCT susceptibility remains unclear. Evidence shows that sperm non-coding RNAs provide a good vision of the environmental stresses experienced by men. Here, to determine whether TGCT impacts the abundance of specific non-coding RNAs in sperm, small RNA deep sequencing analysis of sperm of 25 men aged between 19 and 42 years, diagnosed with (n = 16) or without (n = 9) TGCT was performed. The primary analysis showed no statistical significance in the sncRNA population between the TGCT and non-TGCT groups. However, when sperm physiological parameters were considered to look for differentially expressed sncRNA, we evidenced 11 differentially expressed sncRNA between patients and control which allow a clear discrimination between control and TGCT samples after Hierarchical Clustering analysis. Together, these findings indicate that sperm small non-coding RNAs abundance may have the potential for diagnosing men with TGCT. However, specific care should be taken regarding sperm physiological parameters of the TGCT patients. Hence, larger studies are needed to confirm our findings and to determine whether such a signature associates with the risks to develop TGCT.

The tRNA-derived small RNAs (tsRNAs) are produced in a nuclease-dependent manner in responses to variety of stresses that are common in cancers. We focus on a cancer-enriched tsRNA signature to develop a salivary exosome-based non-invasive biomarker for human esophageal squamous cell carcinoma (ESCC).
Cancer-enriched small RNAs were identified by RNA sequencing of salivary exosomes obtained from ESCC patients (n = 3) and healthy controls (n = 3) in a pilot study and further validated in discovery cohort (n = 66). A multicenter prospective observational study was conducted in two ESCC high-incidence regions (n = 320 and 200, respectively) using the newly developed biomarker signature.
The tsRNA (tRNA-GlyGCC-5) and a previously undocumented small RNA were specifically enriched in salivary exosomes of ESCC patients, ESCC tissues and ESCC cells. The bi-signature composed of these small RNAs was able to discriminate ESCC patients from the controls with high sensitivity (90.50%) and specificity (94.20%). Based on the bi-signature Risk Score for Prognosis (RSP), patients with high-RSP have both shorter overall survival (OS) (HR 4.95, 95%CI 2.90-8.46) and progression-free survival (PFS) (HR 3.69, 95%CI 2.24-6.10) than those with low-RSP. In addition, adjuvant therapy improved OS (HR 0.47, 95%CI 0.29-0.77) and PFS (HR 0.36, 95%CI 0.21-0.62) only for patients with high but not low RSP. These findings are consistent in both training and validation cohort.
The tsRNA-based signature not only has the potential for diagnosis and prognosis but also may serve as a pre-operative biomarker to select patients who would benefit from adjuvant therapy.
A prospective study of diagnosis biomarkers of esophageal squamous cell carcinoma, ChiCTR2000031507 . Registered 3 April 2016 - Retrospectively registered.

Bacterial small RNAs (sRNAs) play a vital role in pathogenesis by enabling rapid, efficient networks of gene attenuation during infection. In recent decades, there has been a surge in the number of proposed and biochemically-confirmed sRNAs in both Gram-positive and Gram-negative pathogens. However, limited homology, network complexity, and condition specificity of sRNA has stunted complete characterization of the activity and regulation of these RNA regulators. To streamline the discovery of the expression of sRNAs, and their post-transcriptional activities, we propose an integrative in vivo data-mining approach that couples DNA protein occupancy, RNA-seq, and RNA accessibility data with motif identification and target prediction algorithms. We benchmark the approach against a subset of well-characterized E. coli sRNAs for which a degree of in vivo transcriptional regulation and post-transcriptional activity has been previously reported, finding support for known regulation in a large proportion of this sRNA set. We showcase the abilities of our method to expand understanding of sRNA RseX, a known envelope stress-linked sRNA for which a cellular role has been elusive due to a lack of native expression detection. Using the presented approach, we identify a small set of putative RseX regulators and targets for experimental investigation. These findings have allowed us to confirm native RseX expression under conditions that eliminate H-NS repression as well as uncover a post-transcriptional role of RseX in fimbrial regulation. Beyond RseX, we uncover 163 putative regulatory DNA-binding protein sites, corresponding to regulation of 62 sRNAs, that could lead to new understanding of sRNA transcription regulation. For 32 sRNAs, we also propose a subset of top targets filtered by engagement of regions that exhibit binding site accessibility behavior in vivo. We broadly anticipate that the proposed approach will be useful for sRNA-reliant network characterization in bacteria. Such investigations under pathogenesis-relevant environmental conditions will enable us to deduce complex rapid-regulation schemes that support infection.

Gene expression regulation is achieved through an intricate network of molecular interactions, in which trans-acting transcription factors (TFs) and small noncoding RNAs (sncRNAs), including microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), play a key role. Recent observations allowed postulating an interplay between TFs and sncRNAs, in that they may possibly share DNA-binding sites. The aim of this study was to analyze the complete subset of miRNA and piRNA sequences stored in the main databases in order to identify the occurrence of conserved motifs and subsequently predict a possible innovative interplay with TFs at a transcriptional level. To this aim, we adopted an original in silico workflow to search motifs and predict interactions within genome-scale regulatory networks. Our results allowed categorizing miRNA and piRNA motifs, with corresponding TFs sharing complementary DNA-binding motifs. The biological interpretation of the gene ontologies of the TFs permitted observing a selective enrichment in developmental pathways, allowing the distribution of miRNA motifs along a topological and chronological frame. In addition, piRNA motifs were categorized for the first time and revealed specific functional implications in somatic tissues. These data might pose experimental hypotheses to be tested in biological models, towards clarifying novel in gene regulatory routes.

The FinO family of proteins constitutes a group of RNA chaperones that interacts with small RNAs (sRNAs) to regulate gene expression in many bacterial species. Here we describe detailed protocols for the biochemical analysis of the RNA chaperone activity of these proteins. Methods are described for preparation of RNA, RNA 5\' end labeling with radioisotope and modified EMSA protocols to test the ability of these proteins to catalyze RNA strand exchange and RNA duplex formation.

Many organisms exchange small RNAs (sRNAs) during their interactions, that can target or bolster defense strategies in host-pathogen systems. Current sRNA-Seq technology can determine the sRNAs present in any symbiotic system, but there are very few bioinformatic tools available to interpret the results. We show that one of the biggest challenges comes from sequences that map equally well to the genomes of both interacting organisms. This arises due to the small size of the sRNAs compared to large genomes, and because a large portion of sequenced sRNAs come from genomic regions that encode highly conserved miRNAs, rRNAs or tRNAs. Here, we present strategies to disentangle sRNA-Seq data from samples of communicating organisms, developed using diverse plant and animal species that are known to receive or exchange RNA with their symbionts. We show that sequence assembly, both de novo and genome-guided, can be used for these sRNA-Seq data, greatly reducing the ambiguity of mapping reads. Even confidently mapped sequences can be misleading, so we further demonstrate the use of differential expression strategies to determine true parasite-derived sRNAs within host cells. We validate our methods on new experiments designed to probe the nature of the extracellular vesicle sRNAs from the parasitic nematode Heligmosomoides bakeri that get into mouse intestinal epithelial cells.

The majority of lung cancer (LC) patients are diagnosed at a late stage, and survival is poor. Circulating RNA molecules are known to have a role in cancer; however, their involvement before diagnosis remains an open question. In this study, we investigated circulating RNA dynamics in prediagnostic LC samples, focusing on smokers, to identify if and when disease-related signals can be detected in serum. We sequenced small RNAs in 542 serum LC samples donated up to 10 years before diagnosis and 519 matched cancer-free controls coming from 905 individuals in the Janus Serum Bank. This sample size provided sufficient statistical power to independently analyze time to diagnosis, stage, and histology. The results showed dynamic changes in differentially expressed circulating RNAs specific to LC histology and stage. The greatest number of differentially expressed RNAs was identified around 7 years before diagnosis for early-stage LC and 1-4 years prior to diagnosis for locally advanced and advanced-stage LC, regardless of LC histology. Furthermore, NSCLC and SCLC histologies have distinct prediagnostic signals. The majority of differentially expressed RNAs were associated with cancer-related pathways. The dynamic RNA signals pinpointed different phases of tumor development over time. Stage-specific RNA profiles may be associated with tumor aggressiveness. Our results improve the molecular understanding of carcinogenesis. They indicate substantial opportunity for screening and improved treatment and will guide further research on early detection of LC. However, the dynamic nature of the RNA signals also suggests challenges for prediagnostic biomarker discovery.

The GacS/A system in Azotobacter vinelandii regulates alginate and alkylresorcinols production through RsmZ1, a small regulatory RNA (sRNA) that releases the translational repression of the algD and arpR mRNAs caused by the RsmA protein. In the Pseudomonadaceae family, the Rsm-sRNAs are grouped into three families: RsmX, RmsY and RsmZ. Besides RsmZ1, A. vinelandii has six other isoforms belonging to the RsmZ family and another one to the RsmY. Environmental signals controlling rsmsRNAs genes in A. vinelandii are unknown. In this work, we present a transcriptional study of the A. vinelandii rsmZ1-7-sRNAs genes, whose transcriptional profiles showed a differential expression pattern, but all of them exhibited their maximal expression at the stationary growth phase. Furthermore, we found that succinate promoted higher expression levels of all the rsmZ1-7 genes compared to glycolytic carbon sources. Single mutants of the rsmZ-sRNAs family were constructed and their impact on alginate production was assessed. We did not observe correlation between the alginate phenotype of each rsmZ-sRNA mutant and the expression level of the corresponding sRNA, which suggests the existence of additional factors affecting their impact on alginate production. Similar results were found in the regulation exerted by the RsmZ-sRNAs on alkylresorcinol synthesis.

Posttranscriptional regulation of gene expression by small noncoding RNAs (sRNAs) is an important control mechanism that modulates bacterial metabolism, motility, and pathogenesis. Using the bacterial carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) system, we here describe an E. coli-based cell-free translation assay that allows a quantitative analysis of translation regulation by ncRNAs and their corresponding translation repressor proteins. The assay quantifies the translation of chloramphenicol acetyltransferase in cell-free expression reactions that contain defined amounts of ncRNA and repressor protein. We demonstrate our protocol with a comparative translation activation analysis of the RsmX, RsmY, and RsmZ sRNAs from Pseudomonas protegens, which reveals a superior efficacy of RsmZ over RsmX and RsmY.