Large-Scale Computational Discovery of Binding Motifs in tRNA Fragments

Accumulating evidence has suggested that tRNA-derived fragments (tRFs) could be loaded to Argonaute proteins and function as regulatory small RNAs. However, their mode of action remains largely unknown, and investigations of their binding mechanisms have been limited, revealing little more than microRNA-like seed regions in a handful of tRFs and a few targets. Here, we identified such regions of potential interaction on a larger scale, using in vivo formed hybrids of guides and targets in crosslinked chimeric reads in two orientations. We considered “forward pairs” (with guides located on the 5′ ends and targets on the 3′ ends of hybrids) and “reverse pairs” (opposite orientation) and compared them as independent sets of biological constructs. We observed intriguing differences between the two chimera orientations, including the paucity of tRNA halves and abundance of polyT-containing targets in forward pairs. We found a total of 197 quality-ranked motifs supported by ∼120,000 tRF–mRNA chimeras, with 103 interacting motifs common in forward and reverse pairs. By analyzing T→C conversions in human and mouse PAR-CLIP datasets, we detected Argonaute crosslinking sites in tRFs, conserved across species. We proposed a novel model connecting the formation of asymmetric pairs in two sets to the potential binding mechanisms of tRFs, involving the identified interaction motifs and crosslinking sites to Argonaute proteins. Our results suggest the way forward for further experimental elucidation of tRF-binding mechanisms.

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MIC-Drop: A platform for large-scale in vivo CRISPR screens

Science ◽

10.1126/science.abi8870 ◽

2021 ◽

pp. eabi8870

Author(s):

Saba Parvez ◽

Chelsea Herdman ◽

Manu Beerens ◽

Korak Chakraborti ◽

Zachary P. Harmer ◽

...

Keyword(s):

Large Scale ◽

Cultured Cells ◽

Cardiac Development ◽

Droplet Microfluidics ◽

Model Organisms ◽

Genetic Screens ◽

Large Numbers ◽

And Function ◽

Genome Scale

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we report Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we show MIC-Drop can identify small molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discover several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse-genetic screens in model organisms.

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Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

10.21203/rs.3.rs-52558/v2 ◽

2020 ◽

Author(s):

R Christian McDonald ◽

Matthew J Schott ◽

Temitope A Idowu ◽

Peter J Lyons

Keyword(s):

Enzyme Activity ◽

Large Scale ◽

Structure And Function ◽

Activation Mechanism ◽

Marker Genes ◽

Synthetic Lethal ◽

Fungal Process ◽

And Function

Abstract Background. Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results. A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions. We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

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Barcoded Rational AAV Vector Evolution enables systematicin vivomapping of peptide binding motifs

10.1101/335372 ◽

2018 ◽

Cited By ~ 2

Author(s):

Marcus Davidsson ◽

Gang Wang ◽

Patrick Aldrin-Kirk ◽

Tiago Cardoso ◽

Sara Nolbrant ◽

...

Keyword(s):

Selection Process ◽

Retrograde Transport ◽

Aav Vector ◽

Binding Motifs ◽

Model Based Clustering ◽

Cell Type Specific ◽

Novel Method ◽

Protein Libraries ◽

And Function

Engineering of Adeno-associated viral (AAV) vector capsids through directed evolution has been used to generate novel capsids with altered tropism and function1-9. This approach, however, involves a selection process that requires multiple generations of screenings to identify real functional capsids2-4. Due to the random nature of this process, it is also inherently unreproducible, and the resulting capsid variants provide little mechanistic insights into the molecular targets engaged. To overcome this, we have developed a novel method for rational capsid evolution named Barcoded Rational AAV Vector Evolution (BRAVE). The key to this method is a novel viral production approach where each virus particle displays a protein-derived peptide on the surface which is linked to a unique barcode in the packaged genome10. Through hidden Markov model-based clustering11, we were able to identify novel consensus motifs for cell-type specific retrograde transport in neurons in vivo in the brain. The BRAVE approach enables the selection of novel capsid structures using only a single-generation screening. Furthermore, it can be used to map, with high resolution, the putative binding sequences of large protein libraries.

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Next Stage Approach to Tissue Engineering Skeletal Muscle

Bioengineering ◽

10.3390/bioengineering7040118 ◽

2020 ◽

Vol 7 (4) ◽

pp. 118

Author(s):

Gregory Reid ◽

Fabio Magarotto ◽

Anna Marsano ◽

Michela Pozzobon

Keyword(s):

Skeletal Muscle ◽

Tissue Engineering ◽

Large Scale ◽

Regeneration Process ◽

Promising Alternative ◽

Alternative Treatment Strategy ◽

Engineered Tissue ◽

And Function ◽

Natural Tissue

Large-scale muscle injury in humans initiates a complex regeneration process, as not only the muscular, but also the vascular and neuro-muscular compartments have to be repaired. Conventional therapeutic strategies often fall short of reaching the desired functional outcome, due to the inherent complexity of natural skeletal muscle. Tissue engineering offers a promising alternative treatment strategy, aiming to achieve an engineered tissue close to natural tissue composition and function, able to induce long-term, functional regeneration after in vivo implantation. This review aims to summarize the latest approaches of tissue engineering skeletal muscle, with specific attention toward fabrication, neuro-angiogenesis, multicellularity and the biochemical cues that adjuvate the regeneration process.

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Application of topic models to a compendium of ChIP-Seq datasets uncovers recurrent transcriptional regulatory modules

Bioinformatics ◽

10.1093/bioinformatics/btz975 ◽

2020 ◽

Vol 36 (8) ◽

pp. 2352-2358

Author(s):

Guodong Yang ◽

Aiqun Ma ◽

Zhaohui S Qin ◽

Li Chen

Keyword(s):

Cell Lines ◽

Large Scale ◽

Topic Models ◽

R Package ◽

Potential Interaction ◽

Computational Method ◽

Supplementary Information ◽

Regulatory Module ◽

Transcriptional Regulatory

Abstract Motivation The availability of thousands of genome-wide coupling chromatin immunoprecipitation (ChIP)-Seq datasets across hundreds of transcription factors (TFs) and cell lines provides an unprecedented opportunity to jointly analyze large-scale TF-binding in vivo, making possible the discovery of the potential interaction and cooperation among different TFs. The interacted and cooperated TFs can potentially form a transcriptional regulatory module (TRM) (e.g. co-binding TFs), which helps decipher the combinatorial regulatory mechanisms. Results We develop a computational method tfLDA to apply state-of-the-art topic models to multiple ChIP-Seq datasets to decipher the combinatorial binding events of multiple TFs. tfLDA is able to learn high-order combinatorial binding patterns of TFs from multiple ChIP-Seq profiles, interpret and visualize the combinatorial patterns. We apply the tfLDA to two cell lines with a rich collection of TFs and identify combinatorial binding patterns that show well-known TRMs and related TF co-binding events. Availability and implementation A software R package tfLDA is freely available at https://github.com/lichen-lab/tfLDA. Supplementary information Supplementary data are available at Bioinformatics online.

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Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

BMC Molecular and Cell Biology ◽

10.1186/s12860-020-00330-w ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

R. Christian McDonald ◽

Matthew J. Schott ◽

Temitope A. Idowu ◽

Peter J. Lyons

Keyword(s):

Enzyme Activity ◽

Large Scale ◽

Structure And Function ◽

Activation Mechanism ◽

Marker Genes ◽

Synthetic Lethal ◽

Fungal Process ◽

And Function

Abstract Background Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

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Research frontiers of chemical detection and measurements

Pure and Applied Chemistry ◽

10.1515/pac-2020-1008 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Zhi-Yuan Gu ◽

Shi-Shu Yang ◽

Ming Xu ◽

Xiang-Da Zhang ◽

Chunhai Fan ◽

...

Keyword(s):

Large Scale ◽

Scientific Instrument ◽

Chemical Detection ◽

Resolution Limit ◽

Chemical Research ◽

Public Health Screening ◽

The Future ◽

Electron Transfer Processes ◽

And Function

Abstract Over the past decade, the global research agenda focused on finding paths to the future development of chemistry. Chemical detection and measurement is an important and fundamental discipline of chemistry and it keeps playing a significant role in chemical research in the next 10–15 years. The goal of chemical detection is to overcome the state-of-art temporospatial resolution limit. Meanwhile, the application of modern chemical technology to serve human health is also highly concerned. We have summarized 10 aspects related to the field, including complex samples analysis, biomacromolecule structure and function, single-entity analysis, large-scale scientific instrument, rapid chemical and electron transfer processes, big data and artificial intelligence, bioimaging, in vivo real-time detection, biomolecule recognition and disease therapy, and public health screening. In this review, we outlined the history and the frontiers of chemical detection and measurements in enhancing the development of chemical sciences. Moreover, we also focused on the major challenges that should be solved in the future in which researchers should develop new techniques and analytical methods to drastically accelerate chemical sciences.

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Basophils Orchestrating Eosinophils’ Chemotaxis and Function in Allergic Inflammation

Cells ◽

10.3390/cells10040895 ◽

2021 ◽

Vol 10 (4) ◽

pp. 895

Author(s):

Joseena Iype ◽

Michaela Fux

Keyword(s):

Current Knowledge ◽

Allergic Inflammation ◽

Potential Interaction ◽

Cytokines And Chemokines ◽

New Findings ◽

Th2 Immunity ◽

Experimental Allergy ◽

And Function ◽

Inflammatory Tissue

Eosinophils are well known to contribute significantly to Th2 immunity, such as allergic inflammations. Although basophils have often not been considered in the pathogenicity of allergic dermatitis and asthma, their role in Th2 immunity has become apparent in recent years. Eosinophils and basophils are present at sites of allergic inflammations. It is therefore reasonable to speculate that these two types of granulocytes interact in vivo. In various experimental allergy models, basophils and eosinophils appear to be closely linked by directly or indirectly influencing each other since they are responsive to similar cytokines and chemokines. Indeed, basophils are shown to be the gatekeepers that are capable of regulating eosinophil entry into inflammatory tissue sites through activation-induced interactions with endothelium. However, the direct evidence that eosinophils and basophils interact is still rarely described. Nevertheless, new findings on the regulation and function of eosinophils and basophils biology reported in the last 25 years have shed some light on their potential interaction. This review will focus on the current knowledge that basophils may regulate the biology of eosinophil in atopic dermatitis and allergic asthma.

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Genome-Wide Identification of CBX2 Targets: Insights in the Human Sex Development Network

Molecular Endocrinology ◽

10.1210/me.2014-1339 ◽

2015 ◽

Vol 29 (2) ◽

pp. 247-257 ◽

Cited By ~ 26

Author(s):

Wassim Eid ◽

Lennart Opitz ◽

Anna Biason-Lauber

Keyword(s):

Candidate Genes ◽

Large Scale ◽

Disorders Of Sex Development ◽

Human Patient ◽

Sex Development ◽

Genome Wide ◽

Identification Technique ◽

And Function ◽

Development Network

Abstract Chromobox homolog 2 (CBX2) is a chromatin modifier that plays an important role in sexual development and its disorders (disorders of sex development [DSD]), yet the exact rank and function of human CBX2 in this pathway remains unclear. Here, we performed large-scale mapping and analysis of in vivo target loci of the protein CBX2 in Sertoli-like NT-2D1 cells, using the DNA adenine methyltransferase identification technique. We identified close to 1600 direct targets for CBX2. Intriguingly, validation of selected candidate genes using qRT-PCR in cells overexpressing CBX2 or in which CBX2 has been knocked down indicated that several CBX2-responsive genes encode proteins that are involved in DSD. We further validated these effects on the candidate genes using a mutated CBX2 causing DSD in human patient. Overall, our findings suggest that CBX2 role in the sex development cascade is to stimulate the male pathway and concurrently inhibit the female pathway. These data provide fundamental insights into potential etiology of DSD.

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Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

10.21203/rs.3.rs-52558/v1 ◽

2020 ◽

Author(s):

R Christian McDonald ◽

Matthew J Schott ◽

Temitope A Idowu ◽

Peter J Lyons

Keyword(s):

Enzyme Activity ◽

Large Scale ◽

Structure And Function ◽

Activation Mechanism ◽

Marker Genes ◽

Synthetic Lethal ◽

Fungal Process ◽

And Function

Abstract Background. Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast S. cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results. A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, his6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14. Conclusions. We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

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