identification-functionally-relevant-lentivirus-integration-sites-an

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens ...

huaqiao university

Livestock-associated  colonization and/or infections exist in pigs and people in frequent contact with pigs. In this study, a total of 130  isolates obtained from different stages of pork production were subjected to antimicrobial susceptibility, biofilm formation, as well as PCR screening to identify virulence genes, and the accessory gene regulator alleles (). Among all 130  isolates, 109 (83.8%, 109/130) isolates were positive for . All swine farms isolates belonged to  IV, whereas  isolated from slaughterhouse and retail indicated diverse  types. All isolates exhibited biofilm formation ability, and raw meat isolates (belonging to  I) exhibited a greater ability to form strong biofilms than swine farms isolates (belonging to  IV). -positive isolates were associated with more virulence genes than -negative isolates. Most biofilm-producing isolates were positive for microbial surface component recognizing adhesive matrix molecule (MSCRAMM), capsule type and  group genes. The results illustrate a significant association between the prevalence rate of MSCRAMM, capsule type and  group genes among isolates producing weak, moderate and strong biofilms. The high prevalence of resistance to ciprofloxacin, gentamicin, tetracycline, clarithromycin, clindamycin, and trimethoprim-sulfamethoxazole were mainly observed in moderate and weak biofilm producers. Our findings indicate that  isolates from pork production displayed diverse molecular ecology.

Association Between  Type, Virulence Factors, Biofilm Formation and Antibiotic Resistance of  Isolates From Pork Production.

Single-strand breaks (SSBs) represent the major form of DNA damage, yet techniques to map these lesions genome-wide with nucleotide-level precision are limited. Here, we present a method, termed SSiNGLe, and demonstrate its utility to explore the distribution and dynamic changes in genome-wide SSBs in response to different biological and environmental stimuli. We validate SSiNGLe using two very distinct sequencing techniques and apply it to derive global profiles of SSBs in different biological states. Strikingly, we show that patterns of SSBs in the genome are non-random, specific to different biological states, enriched in regulatory elements, exons, introns, specific types of repeats and exhibit differential preference for the template strand between exons and introns. Furthermore, we show that breaks likely contribute to naturally occurring sequence variants. Finally, we demonstrate strong links between SSB patterns and age. Overall, SSiNGLe provides access to unexplored realms of cellular biology, not obtainable with current approaches.

Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells.

Long non-coding (lnc) RNAs represent a fascinating class of transcripts that remains highly controversial mainly due to ambiguity surrounding overall biological relevance of these RNAs. Multitude of reverse genetics studies showing functionality of lncRNAs are unfortunately based on assays that are either plagued by non-specific effects and/or cannot unambiguously assign observed phenotypes to the transcript per se. Here, we show application of the novel CRISPR/Cas13 RNA knockdown system that has superior specificity compared to other transcript-targeting knockdown methods like RNAi. We applied this method to a novel widespread subclass of nuclear lncRNAs - very long intergenic non-coding (vlinc) RNAs - in a high-throughput phenotypic assay based on survival challenge in response to anticancer drug treatments. We used multiple layers of controls including mismatch control for each targeting gRNA to ensure uncovering true phenotype-transcript relationships. We found evidence supporting importance for cellular survival for up to 60% of the tested protein-coding mRNAs and, importantly, 64% of vlincRNAs. Overall, this study demonstrates utility of CRISPR/Cas13 as a highly sensitive and specific tool for reverse genetics study of both protein-coding genes and lncRNAs. Furthermore, importantly, this approach provides evidence supporting biological significance of the latter transcripts in anticancer drug response.

A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response.

Long non-coding RNAs (lncRNAs) represent a major fraction of the transcriptome in multicellular organisms. Although a handful of well-studied lncRNAs are broadly recognized as biologically meaningful, the fraction of such transcripts out of the entire collection of lncRNAs remains a subject of vigorous debate. Here we review the evidence for and against biological functionalities of lncRNAs and attempt to arrive at potential modes of lncRNA functionality that would reconcile the contradictory conclusions. Finally, we discuss different strategies of phenotypic analyses that could be used to investigate such modes of lncRNA functionality.

Reverse-genetics studies of lncRNAs-what we have learnt and paths forward.

Although naturally occurring catalytic RNA molecules-ribozymes-have attracted a great deal of research interest, very few have been identified in humans. Here, we developed a genome-wide approach to discovering self-cleaving ribozymes and identified a naturally occurring ribozyme in humans. The secondary structure and biochemical properties of this ribozyme indicate that it belongs to an unidentified class of small, self-cleaving ribozymes. The sequence of the ribozyme exhibits a clear evolutionary path, from its appearance between ~130 and ~65 million years ago (Ma), to acquiring self-cleavage activity very recently, ~13-10 Ma, in the common ancestors of humans, chimpanzees and gorillas. The ribozyme appears to be functional in vivo and is embedded within a long noncoding RNA belonging to a class of very long intergenic noncoding RNAs. The presence of a catalytic RNA enzyme in lncRNA creates the possibility that these transcripts could function by carrying catalytic RNA domains.

Hovlinc is a recently evolved class of ribozyme found in human lncRNA.

The majority of the human genome is transcribed in the form of long non-coding (lnc) RNAs. While these transcripts have attracted considerable interest, their molecular mechanisms of function and biological significance remain controversial. One of the main reasons behind this lies in the significant challenges posed by lncRNAs requiring the development of novel methods and concepts to unravel their functionality. Existing methods often lack cross-validation and independent confirmation by different methodologies and therefore leave significant ambiguity as to the authenticity of the outcomes. Nonetheless, despite all the caveats, it appears that lncRNAs may function, at least in part, by regulating other genes via chromatin interactions. Therefore, the function of a lncRNA could be inferred from the function of genes it regulates. In this work, we present a genome-wide functional annotation strategy for lncRNAs based on identification of their regulatory networks via the integration of three distinct types of approaches: co-expression analysis, mapping of lncRNA-chromatin interactions, and assaying molecular effects of lncRNA knockdowns obtained using an inducible and highly specific CRISPR/Cas13 system.

Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans.

Single-strand breaks (SSBs) represent one of the most common types of DNA damage, yet not much is known about the genome landscapes of this type of DNA lesions in mammalian cells. Here, we found that SSBs are more likely to occur in certain positions of the human genome-SSB hotspots-in different cells of the same cell type and in different cell types. We hypothesize that the hotspots are likely to represent biologically relevant breaks. Furthermore, we found that the hotspots had a prominent tendency to be enriched in the immediate vicinity of transcriptional start sites (TSSs). We show that these hotspots are not likely to represent technical artifacts or be caused by common mechanisms previously found to cause DNA cleavage at promoters, such as apoptotic DNA fragmentation or topoisomerase type II (TOP2) activity. Therefore, such TSS-associated hotspots could potentially be generated using a novel mechanism that could involve preferential cleavage at cytosines, and their existence is consistent with recent studies suggesting a complex relationship between DNA damage and regulation of gene expression.

Hotspots of single-strand DNA "breakome" are enriched at transcriptional start sites of genes.

Studies using highly sensitive targeted RNA enrichment methods have shown that a large portion of the human transcriptome remains to be discovered and that most of the genome is transcribed in a complex, interleaved fashion characterized by a complex web of transcripts emanating from protein coding and noncoding loci. These results resonate with those from single-cell transcriptome profiling endeavors that reveal the existence of multiple novel, cell type-specific transcripts and clearly demonstrate that our understanding of the complexities of the human transcriptome is far from being complete. Here, we review the current status of the targeted RNA enrichment techniques, their application to the discovery of novel cell type-specific transcripts, and their impact on our understanding of the human genome and transcriptome.

Complexities of mammalian transcriptome revealed by targeted RNA enrichment techniques.

The proper replication of mitochondrial DNA is key to the maintenance of this crucial organelle. Multiple studies aimed at understanding the mechanisms of replication of the mitochondrial genome have been conducted in the past several decades; however, while highly informative, they were conducted using relatively low-sensitivity techniques. Here, we established a high-throughput approach based on next-generation sequencing to identify replication start sites with nucleotide-level resolution and applied it to the genome of mitochondria from different human and mouse cell types. We found complex and highly reproducible patterns of mitochondrial initiation sites, both previously annotated and newly discovered in this work, that showed differences among different cell types and species. These results suggest that the patterns of the replication initiation sites are dynamic and might reflect, in some yet unknown ways, the complexities of mitochondrial and cellular physiology. Overall, this work suggests that much remains unknown about the details of mitochondrial DNA replication in different biological states, and the method established here opens up a new avenue in the study of the replication of mitochondrial and potentially other genomes.

Dynamic Patterns of Mammalian Mitochondrial DNA Replication Uncovered Using SSiNGLe-5'ES.

Conversion or editing of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common post-transcriptional RNA modifications with emerging connection to disease. A-to-I conversions can happen at specific sites and lead to increase in proteome diversity and changes in RNA stability, splicing, and regulation. Such sites can be detected as adenine-to-guanine sequence changes by next-generation RNA sequencing which resulted in millions reported sites from multiple genome-wide surveys. Nonetheless, the lack of extensive independent validation in such endeavors, which is critical considering the relatively high error rate of next-generation sequencing, leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them.

Lessons from discovery of true ADAR RNA editing sites in a human cell line.

Endogenous single-stranded DNA (essDNA) can form in a mammalian genome as the result of a variety of molecular processes and can both play important roles inside the cell as well as have detrimental consequences to genome integrity, much of which remains to be fully understood. Here, we established the SSiNGLe-P1 approach based on limited digestion by P1 endonuclease for high-throughput genome-wide identification of essDNA regions. We applied this method to profile essDNA in both human mitochondrial and nuclear genomes. In the mitochondrial genome, the profiles of essDNA provide new evidence to support the strand-displacement model of mitochondrial DNA replication. In the nuclear genome, essDNA regions were found to be enriched in certain types of functional genomic elements, particularly, the origins of DNA replication, R-loops, and to a lesser degree, in promoters. Furthermore, interestingly, many of the essDNA regions identified by SSiNGLe-P1 have not been annotated and thus could represent yet unknown functional elements.

Genome-Wide Profiling of Endogenous Single-Stranded DNA Using the SSiNGLe-P1 Method.

Fraction of functional sequence in the human genome remains a key unresolved question in Biology and the subject of vigorous debate. While a plethora of studies have connected a significant fraction of human DNA to various biochemical processes, the classical definition of function requires evidence of effects on cellular or organismal fitness that such studies do not provide. Although multiple high-throughput reverse genetics screens have been developed to address this issue, they are limited to annotated genomic elements and suffer from non-specific effects, arguing for a strong need to develop additional functional genomics approaches.

Evidence for widespread existence of functional novel and non-canonical human transcripts.

Accurate and comprehensive genomic annotation, including the full list of protein-coding genes, is vital for understanding the molecular mechanisms of human biology. We have previously shown that the genome contains a multitude of yet hidden functional exons and transcripts, some of which might represent novel mRNAs. These results resonate with those from other groups and strongly argue that two decades after the completion of the first draft of the human genome sequence, the current annotation of human genes and transcripts remains far from being complete.

Article	Year
Association Between Type, Virulence Factors, Biofilm Formation and Antibiotic Resistance of Isolates From Pork Production. Frontiers in microbiology\| PubMed ID: 30177917	2018
Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells. Nature communications\| PubMed ID: 31862872	2019
A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response. Scientific reports\| PubMed ID: 32020014	2020
Reverse-genetics studies of lncRNAs-what we have learnt and paths forward. Genome biology\| PubMed ID: 32290841	2020
Hovlinc is a recently evolved class of ribozyme found in human lncRNA. Nature chemical biology\| PubMed ID: 33753927	2021
Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans. BMC biology\| PubMed ID: 34016118	2021
Hotspots of single-strand DNA "breakome" are enriched at transcriptional start sites of genes. Frontiers in molecular biosciences\| PubMed ID: 36046604	2022
Complexities of mammalian transcriptome revealed by targeted RNA enrichment techniques. Trends in genetics : TIG\| PubMed ID: 36681580	2023
Dynamic Patterns of Mammalian Mitochondrial DNA Replication Uncovered Using SSiNGLe-5'ES. International journal of molecular sciences\| PubMed ID: 37298662	2023
Lessons from discovery of true ADAR RNA editing sites in a human cell line. BMC biology\| PubMed ID: 37468903	2023
Genome-Wide Profiling of Endogenous Single-Stranded DNA Using the SSiNGLe-P1 Method. International journal of molecular sciences\| PubMed ID: 37569439	2023
Evidence for widespread existence of functional novel and non-canonical human transcripts. BMC biology\| PubMed ID: 38001496	2023
A novel human protein-coding locus identified using a targeted RNA enrichment technique. BMC biology\| PubMed ID: 39593153	2024

Dongyang Xu

Biography

JoVE Journal Publications

Other Publications