Noncoding Segments Research Articles

LET-7a and LET-7b as Potential Signatures for Glioma Recurrence, Regardless of WHO Tumor Grade.

Micro RNAs (miRs; miRNAs) are small, non-coding RNA segments that influence gene expression. To do this, they bind to corresponding mRNA segments and inhibit the subsequent protein translation. Currently, about 200 transcripts are thought to be regulated by a single miRNA. Dysregulated miRNAs can act as tumor promoters as well as tumor suppressors. The aim of this study was to verify miR-LET-7a and miR-LET-7b as a signature for glioma recurrence detection by analyzing their expression in glioma tissue. Only patients with two or more recurrences of glioma (n=25) were included in the study (tissue samples, n=89). Tissue was obtained during neurosurgical procedures and shock-frozen in liquid nitrogen. The patient cohort (female:male=12:13) had an average age of 35 years when first diagnosed (primary tumor: 13 WHO grade II, 7 WHO grade III and 5 WHO grade IV) and 48 years in the fifth relapse. Quantitative real-time polymerase chain reaction was performed to analyze miRNA expression. miR-151a-3p was used as an internal reference. Expression of miR-LET-7a and miR-LET-7b was significantly lower in the first recurrence than in the primary tumor. The expression of miR-LET-7a and -7b significantly decreased with increasing tumor grade. The strongest down-regulation was found between WHO grade III and IV. In paired samples in which tumor grade did not change between the primary and the first recurrence, the expression of both miRNAs remained significantly lower in the recurrent than in the primary tumor. miR-LET-7a and miR-LET-7b represent a potential signature for glioma recurrence regardless of WHO grade.

Junk DNA: Is it Really Junk - or a Code Waiting to Be Cracked?

The idea that our genetic material contains Junk DNA is fading. Many DNA segments that were previously believed to be worthless now have important functions, according to recent studies. These non-coding areas, once known as Junk DNA, are crucial for regulating when and how genes function even though they do not directly make proteins. They assist our cells respond to various circumstances and direct the growth of organisms. Certain non-coding DNA sequences function as switches that determine where and when genes should be activated or deactivated. Furthermore, some non-coding DNA segments help maintain the orderly structure of our chromosomes. They also contribute to the evolution of species and may contain remnants of ancient viruses that once infected our forebears. Although scientists still don’t fully understand every aspect of non-coding DNA, it is now recognized as a complex and active element of our biology. Researchers believe that what was once labelled as Junk DNA could actually be very important for our health, development, and ability to adapt to environmental changes. This new perspective is helping us understand how valuable Junk DNA really is.

Chromatin interaction maps of human arterioles reveal new mechanisms for the genetic regulation of blood pressure.

Arterioles are small blood vessels located just upstream of capillaries in nearly all tissues. The constriction and dilation of arterioles regulate tissue perfusion and are primary determinants of systemic blood pressure (BP). Abnormalities in arterioles are central to the development of major diseases such as hypertension, stroke, and microvascular complications of diabetes. Despite the broad and essential role of arterioles in physiology and disease, current knowledge of the functional genomics of arterioles is largely absent, partly because it is challenging to obtain and analyze human arteriole samples. Here, we report extensive maps of chromatin interactions, single-cell expression, and other molecular features in human arterioles and uncover new mechanisms linking human genetic variants to gene expression in vascular cells and the development of hypertension. Compared to large arteries, arterioles exhibited a higher proportion of pericytes which were strongly associated with BP traits. BP-associated single nucleotide polymorphisms (SNPs) were enriched in chromatin interaction regions in arterioles, particularly through enhancer SNP-promoter interactions, which were further linked to gene expression specificity across tissue components and cell types. Using genomic editing in animal models and human induced pluripotent stem cells, we discovered novel mechanisms linking BP-associated noncoding SNP rs1882961 to gene expression through long-range chromatin contacts and revealed remarkable effects of a 4-bp noncoding genomic segment on hypertension in vivo. We anticipate that our rich data and findings will advance the study of the numerous diseases involving arterioles. Moreover, our approach of integrating chromatin interaction mapping in trait-relevant tissues with SNP analysis and in vivo and in vitro genome editing can be applied broadly to bridge the critical gap between genetic discoveries and physiological understanding.

Abstract 45: Deletion of a 4-bp noncoding genomic segment orthologous to human rs1882961 results in sex-divergent changes in salt-induced hypertension in Dahl salt-sensitive rats

Most single nucleotide polymorphisms (SNPs) associated with human blood pressure (BP) are in noncoding regions of the genome and have small effect sizes in the general population. Functional studies in a controlled model system are essential to ascertain the effect of these noncoding loci on blood pressure. Such direct testing is challenging and largely absent, contributing to a critical gap between genetic discoveries and physiological understanding. We reasoned that physiological studies in genetically engineered and phenotypically permissive animal models would help to address this critical gap. We tested the hypothesis by studying the noncoding SNP rs1882961. The T allele of rs1882961, which has a frequency of approximately 30% in the European population, is associated with 0.23 mmHg higher systolic blood pressure in humans. We mapped the human rs1882961 SNP to the rat genome and used CRISPR/Cas9 to delete a 4-bp segment encompassed the rs1882961 orthologous site in the genome of the Dahl salt-sensitive (SS) rat (SS-Δrs1882961). Male SS-Δrs1882961 -/- rats exhibited significantly higher mean arterial pressure (MAP) and systolic and diastolic blood pressures (SBP and DBP) than wild-type littermates after the rats were fed a high-salt (4% NaCl) diet for 14 days (156.6 ± 5.9 vs 149.3 ± 5.1 mmHg for MAP, 179.4 ± 7.8 vs 171.9 ± 6.0 mmHg for SBP, and 133.2 ± 4.6 vs 125.1 ± 4.5 mmHg for DBP; n=7 for SS-Δrs1882961 -/- and 9 for wild-type, p<0.05). In contrast, MAP, SBP, DBP, and pulse pressure of female SS-Δrs1882961 -/- rats were significantly lower than wild-type littermates on a high-salt diet (139.8 ± 1.8 vs 149.5 ± 4.7 mmHg for MAP, 159.3 ± 2.5 vs 172.3 ± 6.0 mmHg for SBP, 116.8 ± 1.5 vs 124.5 ± 3.7 mmHg for DBP, and 42.5 ± 1.6 vs 47.7 ± 2.3 mmHg for pulse pressure; n=8 for SS-Δrs1882961 -/- and 7 for wild-type, p<0.05). The difference in systolic blood pressure reached more than 10 mmHg in female rats. Urinary albumin and protein levels of SS-Δrs1882961 -/- rats after 14 days on a high-salt diet tended to be higher in male and lower in female compared with wild-type littermates. The remarkable effects on BP as well as the sex-divergent changes resulted from a 4-bp noncoding segment deletion around rs1882961 orthologous site indicate that noncoding genomic segment may have profound impact on phenotypes on permissive genomic background and with informative stressors.

CRISPR/Cas9 Technology for Non-Coding Gene Editing in Schizophrenia Therapeutics: The Recent Progress And Challenges

Abstract: Within a decade the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9 system), an advanced gene-editing technology became one of the celebrated approaches in modern disease therapeutics and was reported to have a potential role in the alteration of non-coding RNAs segment which are the pivotal causes behind the several mental disorder such as Schizophrenia. In general, Schizophrenia is referred as a neurodevelopmental disorder and symptomatically exhibited by social deficit, cognitive dysfunction, apathy, delusions, hallucinations, etc. At a genomics level large number of loci are susceptible for genetic alteration in schizophrenia and are mostly located in the genome’s non-coding region. With the growing variants and mutations in ncRNA genes (miRNA and lncRNA) strongly associated with schizophrenia, the need to develop a genetic tool to help with the treatment and study of schizophrenia increases. Recently the use of CRISPR/cas9 technology in the productive alteration of non-coding RNAs genes such as miRNA; miR-291, miR-141, and miR-21, lncRNA, lncRNA-21A, AK023948, and LncRNA Rian has been reported. The Cas9 protein and guide RNA (gRNA) together form the CRISPR/Cas9 system is known to be highly specific and efficient for manipulating the impact of gene mutations linked to genomic DNA like ncRNA besides other inheritable genetic diseases. Copy number variations are also found to be linked with schizophrenia. The generation of reciprocal CNVs of 15q13.3 and 16p11.2 in humaninduced pluripotent stem cells (iPSCs) with the CRISPR/Cas 9 system has opened new possibilities. Still, there are some limitations and challenges yet to be defeated, like the blood-brain barrier poses an obstacle to treating mental disorders and ethical issues like genomic DNA manipulation of eggs and embryos. This review brings schizophrenia-associated ncRNAs and CRISPR gene-editing technology for the non-coding parts of the genomic DNA together and recent challenges.

MiRNA as a Biomarker for the Early Detection of Colorectal Cancer.

MicroRNAs (miRNAs) are short, non-coding RNA segments that can be detected in a variety of clinical samples, including serum, stool, and urine. While miRNAs were initially known for their effect on post-translational gene expression, the last decade of research has shown them to be promising biomarkers for the detection of many types of cancer. This paper explores the use of miRNA detection as a tool for colorectal cancer (CRC) screening. We discuss the current state of miRNA detection, compare it to the existing CRC screening tools, and highlight the advantages and drawbacks of this approach from a clinical and logistical perspective. Our research finds that miRNA-based tests for CRC show great potential, but that widespread clinical adoption will be conditional on future research overcoming key hurdles.

Intron Retention: What that means for the biologist

Introns are non-coding segments within eukaryotic genes that are transcribed along with exons. They are subsequently removed during splicing, and their presence allows for generating diverse mRNA isoforms through the process called alternative splicing. The functional significance of introns going beyond mere "junk DNA" is questionable until recently; scientists identified that these pieces of junk DNA could hang around with the functional mRNA escaping splicing. Intron retention is a type of alternative splicing, a process that occurs during the maturation of messenger RNA (mRNA) in eukaryotic cells. In eukaryotes, genes typically consist of exons (coding regions) and introns (non-coding regions). After transcription, the precursor mRNA (pre-mRNA) undergoes splicing, where introns are removed, and exons are joined together to form the mature mRNA. Intron retention, however, deviates from this typical splicing process. Instead of being entirely removed from the pre-mRNA, the mature mRNA may retain one or more introns. This results in the inclusion of intronic sequences within the final mRNA transcript. The retained introns may contain premature stop codons or regulatory elements that can influence gene expression and protein function. Intron retention is a form of alternative splicing because it generates multiple mRNA isoforms from a single gene. This diversity in mRNA transcripts can produce different protein isoforms with distinct functions or regulatory properties. The regulation of intron retention is complex and involves various cellular factors, including splicing machinery components and RNA-binding proteins. Research on intron retention has revealed its importance in various biological processes, including development, cell growth and differentiation, and diseases like cancer. Aberrant intron retention has been associated with certain genetic disorders and cancers. Understanding the mechanisms and functional consequences of intron retention is crucial for unraveling the complexity of gene regulation and its impact on cellular processes. With an increasing output of data from high throughput assays line RNA Seq, scRNA-Seq, etc., it's high time data scientists look into these signatures in the context of disease pathology. This may well be the tip of the iceberg.

Advancing DNA Signal Processing: Integrating Digital and Biological Nuances for Enhanced Identification of Coding Regions

Within the complex realm of DNA sequencing, discerning protein coding areas from non-coding segments proves challenging due to the pervasive 1/f background disturbance. Traditional digital signal processing (DSP) methodologies, while widely adopted, may inadvertently overlook the inherent nuances and intricacies of DNA sequences. This paper critically examines these established DSP-centric methodologies, underscoring their potential inadequacies in capturing the salient characteristics intrinsic to DNA. Notably, nucleotides within the DNA exhibit distinct attributes, such as their triadic configurations, specific structural significance, and particularized density distributions in codons, among other characteristics. By harnessing these inherent features of nucleotides, computational approaches can effectively counteract signal disruptions, enhancing the precision in identifying protein coding regions.

Abstract 070: Deletion Of A Non-coding Genomic Segment Orthologous To The Human Rs1173771 Haplotype Region Attenuates Hypertension In Dahl Salt-sensitive Rats

Most SNPs associated by GWAS with blood pressure (BP) phenotypes are located in noncoding regions of DNA. Investigating the physiological effects of these SNPs and their underlying mechanisms will advance our understanding of how genetic and epigenetic mechanisms regulate BP. The BP-associated SNP rs1173771 is in linkage disequilibrium (LD) with 10 other SNPs (r 2 >0.80) forming a haplotype spanning 17.5 kb of noncoding DNA. We explored the effects of the rs1173771 LD orthologous region on BP on the genetic background of Dahl salt-sensitive (SS) rats and the physiological and molecular mechanisms involved. A ~29.5 kb noncoding segment on rat chromosome 5 orthologous to the human rs1173771 LD region was identified using LiftOver. The model SS-rs1173771LD -/- was generated using CRISPR-Cas9 to delete this region in SS rats. BP was not significantly different between SS-rs1173771LD -/- rats and wildtype (WT) littermates on a 0.4% NaCl diet. After 14 days of a 4% NaCl diet, 24-hour mean arterial pressure (MAP) and systolic blood pressure (SBP) of SS-rs1173771LD -/- male rats were significantly lower compared with WT (149.1 ± 2.7 vs 155.9 ± 2.5 mmHg for MAP and 169.6 ± 4.3 vs 179.0 ± 3.0 mmHg for SBP). No difference in diastolic blood pressure was observed. No significant difference of BP was observed between genotypes in females. The transcription start sites of human natriuretic peptide receptor 3 (NPR3) and rat Npr3 are located ~129-kbp and ~80-kbp from the human rs1173771 LD region and the rat orthologous region, respectively. RNA-seq indicated that Npr3 expression was upregulated in the mesenteric artery in SS-rs1173771LD -/- rats. Vasodilation of mesenteric arteries in response to C-type natriuretic peptide was significantly enhanced in SS-rs1173771LD -/- male rats compared with WT littermates. A selective NPR-C antagonist abolished vasodilation in both groups, suggesting the vasorelaxation was NPR-C dependent. In conclusion, deletion of the rs1173771 LD orthologous region increased mesenteric artery vasorelaxation and attenuated salt-induced hypertension in male SS rats. Our findings provided evidence that evolutionarily conserved noncoding DNA regions located far from protein-coding genes can influence BP in vivo.

Mesoporous Core-Cone Silica Nanoparticles Can Deliver miRNA-26a to Macrophages to Exert Immunomodulatory Effects on Osteogenesis In Vitro.

Nanoparticles can play valuable roles in delivering nucleic acids, including microRNAs (miRNA), which are small, non-coding RNA segments. In this way, nanoparticles may exert post-transcriptional regulatory influences on various inflammatory conditions and bone disorders. This study used biocompatible, core-cone-structured, mesoporous silica nanoparticles (MSN-CC) to deliver miRNA-26a to macrophages in order to influence osteogenesis in vitro. The loaded nanoparticles (MSN-CC-miRNA-26) showed low-level toxicity towards macrophages (RAW 264.7 cells) and were internalized efficiently, causing the reduced expression of pro-inflammatory cytokines, as seen via real-time PCR and cytokine immunoassays. The conditioned macrophages created a favorable osteoimmune environment for MC3T3-E1 preosteoblasts, driving osteogenic differentiation with enhanced osteogenic marker expression, alkaline phosphatase (ALP) production, extracellular matrix formation, and calcium deposition. An indirect co-culture system revealed that direct osteogenic induction and immunomodulation by MSN-CC-miRNA-26a synergistically increased bone production due to the crosstalk between MSN-CC-miRNA-26a-conditioned macrophages and MSN-CC-miRNA-26a-treated preosteoblasts. These findings demonstrate the value of nanoparticle delivery of miR-NA-26a using MSN-CC for suppressing the production of pro-inflammatory cytokines with macrophages and for driving osteogenic differentiation in preosteoblasts via osteoimmune modulation.

Occurrence, Role, and Challenges of MicroRNA in Human Breast Milk: A Scoping Review

MicroRNAs are non-coding segments of RNA involved in the epigenetic modulation of various biological processes. Their occurrence in biological fluids, such as blood, saliva, tears, and breast milk, has drawn attention to their potential influence on health and disease development. Hundreds of microRNAs have been isolated from breast milk, yet the evidence on their function remains inconsistent and inconclusive. The rationale for the current scoping review is to map the evidence on the occurrence, characterization techniques, and functional roles of microRNAs in breast milk. The review of the sources of this evidence highlights the need to address methodological challenges to achieve future advances in understanding microRNAs in breast milk, particularly their role in conditions such as neoplasms. Nonetheless, remarkable progress has been made in characterizing the microRNA profiles of human breast milk.

Bicuspid Aortic Valve-Associated Regulatory Regions Reveal GATA4 Regulation and Function During Human-Induced Pluripotent Stem Cell-Based Endothelial-Mesenchymal Transition-Brief Report.

The endothelial-mesenchymal transition (EndoMT) is a fundamental process for heart valve formation and defects in EndoMT cause aortic valve abnormalities. Our previous genome-wide association study identified multiple variants in a large chromosome 8 segment as significantly associated with bicuspid aortic valve (BAV). The objective of this study is to determine the biological effects of this large noncoding segment in human induced pluripotent stem cell (hiPSC)-based EndoMT. A large genomic segment enriched for BAV-associated variants was deleted in hiPSCs using 2-step CRISPR/Cas9 editing. To address the effects of the variants on GATA4 expression, we generated CRISPR repression hiPSC lines (CRISPRi) as well as hiPSCs from BAV patients. The resulting hiPSCs were differentiated to mesenchymal/myofibroblast-like cells through cardiovascular-lineage endothelial cells for molecular and cellular analysis. Single-cell RNA sequencing was also performed at different stages of EndoMT induction. The large deletion impaired hiPSC-based EndoMT in multiple biallelic clones compared with their isogenic control. It also reduced GATA4 transcript and protein levels during EndoMT, sparing the other genes nearby the deletion segment. Single-cell trajectory analysis revealed the molecular reprogramming during EndoMT. Putative GATA-binding protein targets during EndoMT were uncovered, including genes implicated in endocardial cushion formation and EndoMT process. Differentiation of cells derived from BAV patients carrying the rs117430032 variant as well as CRISPRi repression of the rs117430032 locus resulted in lower GATA4 expression in a stage-specific manner. TWIST1 was identified as a potential regulator of GATA4 expression, showing specificity to the locus tagged by rs117430032. BAV-associated distal regions regulate GATA4 expression during hiPSC-based EndoMT, which in turn promotes EndoMT progression, implicating its contribution to heart valve development.

Systematically Interrogating Downstream Regulation Mechanisms of HOXA9 in MLL-r Leukemia Via Functional Noncoding CRISPR Screens

HOXA9 overexpression is observed in 50-70% of human acute myeloid leukemia (AML) and a subset of acute lymphoblastic leukemia (ALL) and positively correlates with poor patient outcomes. Leukemia subtypes with hallmark overexpression of HOXA9 include those carrying MLL gene rearrangements (MLL-r), NPM1c mutations, and other genetic alterations. Frequent genetic translation of NUP98-HOXA9 retained the DNA binding domain of HOXA9, suggesting the transcription factor function is essential for leukemia initiation and maintenance. Accumulating evidence indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation. However, HOXA9 protein itself is a poor therapeutic target as it lacks targetable binding domains. Therefore, understanding HOXA9's functional downstream targets will provide alternative therapeutic targets. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory sequences and controls the downstream genes in MLL-r leukemia and across leukemias of other genetic subtypes. In this study, we have successfully established a HOXA9-miniAID expressing MLL-r B-ALL cell line to execute acute HOXA9 degradation in the same isogenic cell line background upon auxin treatment. Compared with conventional loss-of-function strategies, our AID cellular model allows the investigation of immediate downstream targets controlled by HOXA9. We have therefore identified about 1,800 reproducible peaks bound by HOXA9 at a genome-wide scale. About 80% of HOXA9 binding peaks were co-bound by HOXA9's binding partner MEIS1, and motif enrichment confirmed that more than 75% of peaks contained a typical HOXA9 consensus motif. Interestingly, >80% binding peaks are located in non-promoter cis-regulator elements. Given the essential role of HOXA9 in regulating MLL-r leukemia cell survival and differentiation, we reasoned that targeting specific HOXA9-bound peaks will affect downstream gene expression leading to a similar survival crisis seen in HOXA9 knockout cells. Based on this rationale, combinatorial CRISPR screens were performed in three biological replicates on the MLL-r SEM cell line stably expressing Cas9 and base editor (ABE8.0), followed by time-course dropout selection on days 7, 14, and 21. Integrative CRISPR screen analysis identified six reproducible noncoding hits, including a positive control located in the distal enhancer of FLT3. Functional validation by targeting these six noncoding segments with Cas9, dCas9-KRAB in additional MLL-r leukemia cell lines MOLM13, MV4,11 confirmed the survival essential of these HOXA9-bound regions. No cellular phenotype was observed in non-MLL-r leukemia cell lines, including NALM6, JURKAT, or OCI-AML2. Transplanting SEM cells targeted with sgHOXA9-in-FLT3 into NSG mice, significant growth retardation was monitored by flow analysis of peripheral blood. In addition, RNA-seq and Q-PCR analysis further identified the functional relevant downstream genes upon CRISPR editing of these HOXA9-bound noncoding segments, including FLT3, XBP1, JUN, BAHCC1, and CCDC200. Chromatin conformation characterization by Capture-C further confirmed the long-range interaction between HOXA9 bound enhancers and corresponding gene promoters in MLL-r SEM cells. In summary, using state-of-art research tools and newly established cell models, we have conducted a systematic functional screen and mechanism study of an undocumented downstream transcriptional network of HOXA9 in MLL-r leukemia cells. The work is significant as it will advance our understanding of how HOXA9-associated transcription programs reconstruct the regulatory network specifying MLL-r dependency. Moreover, our study will promote the future development of alternative therapeutic targets in HOXA9-driven (including HOXA9 and possibly NUP98-HOXA9 subtype) leukemia in patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Typical Enhancers, Super-Enhancers, and Cancers.

Simple SummaryThe cancer genome has been exhaustively studied upon the advent of Next-Generation Sequencing technologies. Coding and non-coding sequences have been defined as hotspots of genomic variations that affect the naïve gene expression programs established in normal cells, thus working as endogenous drivers of carcinogenesis. In this review, we comprehensively summarize fundamental aspects of gene expression regulation, with emphasis on the impact of sequence and structural variations mapped across non-coding cis-acting elements of genes encoding for tumor-related transcription factors. Chromatin architecture, epigenome reprogramming, transcriptional enhancers and Super-enhancers, oncogene regulation, cutting-edge technologies, and pharmacological treatment are substantially highlighted. Non-coding segments of the human genome are enriched in cis-regulatory modules that constitute functional elements, such as transcriptional enhancers and Super-enhancers. A hallmark of cancer pathogenesis is the dramatic dysregulation of the “archetype” gene expression profiles of normal human cells. Genomic variations can promote such deficiencies when occurring across enhancers and Super-enhancers, since they affect their mechanistic principles, their functional capacity and specificity, and the epigenomic features of the chromatin microenvironment across which these regulatory elements reside. Here, we comprehensively describe: fundamental mechanisms of gene expression dysregulation in cancers that involve genomic abnormalities within enhancers’ and Super-enhancers’ (SEs) sequences, which alter the expression of oncogenic transcription factors (TFs); cutting-edge technologies applied for the analysis of variation-enriched hotspots of the cancer genome; and pharmacological approaches for the treatment of Super-enhancers’ aberrant function. Finally, we provide an intratumor meta-analysis, which highlights that genomic variations in transcription-factor-driven tumors are accompanied overexpression of genes, a portion of which encodes for additional cancer-related transcription factors.

PmiRtarbase: A positive miRNA-target regulations database

MicroRNAs (miRNAs) are ~22 nt small non-coding RNA segments that are widely involved in the regulation of gene expression. Accumulating evidences show that miRNAs not only inhibit the expression of some targeted genes but also promote that of some targeted genes in specific conditions. Over the past decades, many miRNA-target databases have been developed from computational prediction and/or experimental validation perspectives. However, there is no database available to systematically collect positive miRNA-target associations that are essential in deciphering the miRNA regulation mechanism. To promote the miRNA study, we developed a new database: PmiRtarbase that acquires validated positive miRNA-target interactions by mining published literature. It includes 312 curated associations between 119 miRNAs and 169 genes in 8 species from 130 studies and summarizes the conditions and detailed descriptions of the miRNA-target associations. We also constructed a database named PmiRtarbase, a user-friendly interface to conveniently search and download all related entries. This elaborate database aims to serve as a beneficial resource for studying the miRNA positive regulation mechanism and miRNA-based therapeutics. Data availabilityThe full positive miRNA-target data can be accessed through the link http://www.lwb-lab.cn/PmiRtarbase. Users of this dataset should acknowledge the contributions of the original authors and properly cite this article.

Whole Genome Sequence Analysis of a Prototype Strain of the Novel Putative Rotavirus Species L.

Rotaviruses infect humans and animals and are a main cause of diarrhea. They are non-enveloped viruses with a genome of 11 double-stranded RNA segments. Based on genome analysis and amino acid sequence identities of the capsid protein VP6, the rotavirus species A to J (RVA-RVJ) have been defined so far. In addition, rotaviruses putatively assigned to the novel rotavirus species K (RVK) and L (RVL) have been recently identified in common shrews (Sorex araneus), based on partial genome sequences. Here, the complete genome sequence of strain KS14/0241, a prototype strain of RVL, is presented. The deduced amino acid sequence for VP6 of this strain shows only up to 47% identity to that of RVA to RVJ reference strains. Phylogenetic analyses indicate a clustering separated from the established rotavirus species for all 11 genome segments of RVL, with the closest relationship to RVH and RVJ within the phylogenetic RVB-like clade. The non-coding genome segment termini of RVL showed conserved sequences at the 5′-end (positive-sense RNA strand), which are common to all rotaviruses, and those conserved among the RVB-like clade at the 3′-end. The results are consistent with a classification of the virus into a novel rotavirus species L.

Genetic and biological characteristics of species A rotaviruses detected in common shrews suggest a distinct evolutionary trajectory.

Species A rotaviruses (RVAs) are important aetiological agents of severe diarrhoea in young children. They are also widely distributed in mammals and birds, and increasing evidence indicates the possibility of zoonotic transmission of RVA strains between animals and humans. Moreover, reassortment of the eleven segments of the RVA genome can result in rapid biological changes and may influence pathogenic properties. Here, the nearly complete genome of an RVA strain from a common shrew (Sorex araneus) was sequenced, which showed high nucleotide sequence similarity to additionally determined partial sequences from common shrew RVAs but only very low identity (below 68 per cent) to RVAs from other animal species and humans. New genotypes were assigned to most genome segments of the novel common shrew RVA strain KS14/269, resulting in the genome constellation G39-P[55]-I27-R26-C22-M22-A37-N26-T26-E30-H26. Phylogenetic analyses clustered the common shrew RVAs as ancestral branches of other mammalian and avian RVAs for most of the genome segments, which is in contrast to the phylogeny of the hosts. Nevertheless, conserved sequences typical for all RVAs were identified at the 5ʹ- and 3ʹ- non-coding segment termini. To explore whether the common shrew RVA can exchange genetic material with other mammalian RVAs by reassortment, a reverse genetics system based on the simian RVA strain SA11 was used. However, no viable reassortants could be rescued by exchanging the VP4-, VP6-, or VP7-encoding genome segment alone or in combinations. It can be concluded that highly divergent RVAs are present in common shrews, indicating an evolution of these viruses largely separated from other mammalian and avian RVAs. The zoonotic potential of the virus seems to be low but needs to be further analysed in future.

74. STRUCTURAL EQUATION MODELLING IN THE GENOMICS ERA: STATE-OF-THE-ART AND FUTURE DEVELOPMENTS

Some genera of ciliates, such as Oxytricha and Stylonychia, undergo massive genome reorganization during development and provide model organisms to study DNA rearrangement. A common feature of these ciliates is the presence of two types of nuclei: a germline micronucleus and a transcriptionally-active somatic macronucleus containing over 16,000 gene sized “nano-chromosomes”. During conjugation the old parental macronucleus disintegrates and a new macronucleus forms from a copy of the zygotic micronucleus. During this process, macronuclear chromosomes assemble through DNA processing events that delete 90–98% of the DNA content of the micronucleus. This includes the deletion of noncoding DNA segments that interrupt precursor DNA regions in the micronucleus, as well as transposons and other germline-limited DNA. Each macronuclear locus may be present in the micronucleus as several nonconsecutive, permuted, and/or inverted DNA segments. Here we investigate the genome-wide range of scrambled gene architectures that describe all precursor-product relationships in Oxytricha trifallax, the first completely sequenced scrambled genome. We find that five general, recurrent patterns in the sets of scrambled micronuclear precursor pieces can describe over 80% of Oxytricha's scrambled genes. These include instances of translocations and inversions, and other specific patterns characterized by alternating stretches of consecutive odd and even DNA segments. Moreover, we find that iterating patterns of alternating odd–even segments up to four times can describe over 96% of the scrambled precursor loci. Recurrence of these highly structured genetic architectures within scrambled genes presumably reflects recurrent evolutionary events that gave rise to over 3000 of scrambled loci in the germline genome.

Pervasive transcription of the mitochondrial genome in Candida albicans is revealed in mutants lacking the mtEXO RNase complex

ABSTRACT The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3ʹ-5ʹ exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wild-type strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense (‘mirror’) transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments.

Next-Generation Sequencing Identifies Extended HLA Class I and II Haplotypes Associated With Early-Onset and Late-Onset Myasthenia Gravis in Italian, Norwegian, and Swedish Populations.

Genetic susceptibility to myasthenia gravis (MG) associates with specific HLA alleles and haplotypes at the class I and II regions in various populations. Previous studies have only examined alleles at a limited number of HLA loci that defined only broad serotypes or alleles defined at the protein sequence level. Consequently, genetic variants in noncoding and untranslated HLA gene segments have not been fully explored but could also be important determinants for MG. To gain further insight into the role of HLA in MG, we applied next-generation sequencing to analyze sequence variation at eleven HLA genes in early-onset (EO) and late-onset (LO) non-thymomatous MG patients positive for the acetylcholine receptor (AChR) antibodies and ethnically matched controls from Italy, Norway, and Sweden. For all three populations, alleles and haplotype blocks present on the ancestral haplotype AH8.1 were associated with risk in AChR-EOMG patients. HLA-B*08:01:01:01 was the dominant risk allele in Italians (OR = 3.28, P = 1.83E−05), Norwegians (OR = 3.52, P = 4.41E−16), and in Swedes HLA-B*08:01 was the primary risk allele (OR = 4.24, P <2.2E-16). Protective alleles and haplotype blocks were identified on the HLA-DRB7, and HLA-DRB13.1 class II haplotypes in Italians and Norwegians, whereas in Swedes HLA-DRB7 exhibited the main protective effect. For AChR-LOMG patients, the HLA-DRB15.1 haplotype and associated alleles were significantly associated with susceptibility in all groups. The HLA-DR13–HLA-DR–HLA-DQ haplotype was associated with protection in all AChR-LOMG groups. This study has confirmed and extended previous findings that the immunogenetic predisposition profiles for EOMG and LOMG are distinct. In addition, the results are consistent with a role for non-coding HLA genetic variants in the pathogenesis of MG.