Structure and functional implications of WYL-domain-containing transcription factor PafBC involved in the mycobacterial DNA damage response

AbstractIn mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determined the crystal structure ofArthrobacter aurescensPafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA molecules. Using anin vivomutational screen inMycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.

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Structure and functional implications of WYL domain-containing bacterial DNA damage response regulator PafBC

Nature Communications ◽

10.1038/s41467-019-12567-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Andreas U. Müller ◽

Marc Leibundgut ◽

Nenad Ban ◽

Eilika Weber-Ban

Keyword(s):

Dna Damage ◽

Transcription Factors ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Information ◽

Response Regulator ◽

Bacterial Dna ◽

Bacterial Transcription ◽

Arthrobacter Aurescens

Abstract In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determine the crystal structure of Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA-binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA-binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA or other nucleic acid molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.

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The Emerging Role and Promise of Circular RNAs in Obesity and Related Metabolic Disorders

Cells ◽

10.3390/cells9061473 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1473

Author(s):

Mohamed Zaiou

Keyword(s):

Rna Binding ◽

Metabolic Diseases ◽

Rna Binding Proteins ◽

In Vivo Studies ◽

Future Research ◽

Circular Rnas ◽

Exciting Field ◽

Rna Molecules

Circular RNAs (circRNAs) are genome transcripts that are produced from back-splicing of specific regions of pre-mRNA. These single-stranded RNA molecules are widely expressed across diverse phyla and many of them are stable and evolutionary conserved between species. Growing evidence suggests that many circRNAs function as master regulators of gene expression by influencing both transcription and translation processes. Mechanistically, circRNAs are predicted to act as endogenous microRNA (miRNA) sponges, interact with functional RNA-binding proteins (RBPs), and associate with elements of the transcriptional machinery in the nucleus. Evidence is mounting that dysregulation of circRNAs is closely related to the occurrence of a range of diseases including cancer and metabolic diseases. Indeed, there are several reports implicating circRNAs in cardiovascular diseases (CVD), diabetes, hypertension, and atherosclerosis. However, there is very little research addressing the potential role of these RNA transcripts in the occurrence and development of obesity. Emerging data from in vitro and in vivo studies suggest that circRNAs are novel players in adipogenesis, white adipose browning, obesity, obesity-induced inflammation, and insulin resistance. This study explores the current state of knowledge on circRNAs regulating molecular processes associated with adipogenesis and obesity, highlights some of the challenges encountered while studying circRNAs and suggests some perspectives for future research directions in this exciting field of study.

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The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity

Life Science Alliance ◽

10.26508/lsa.201800187 ◽

2018 ◽

Vol 1 (5) ◽

pp. e201800187 ◽

Cited By ~ 8

Author(s):

Daniela Lazzaretti ◽

Lina Bandholz-Cajamarca ◽

Christiane Emmerich ◽

Kristina Schaaf ◽

Claire Basquin ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Information ◽

Target Selection ◽

Mrna Localization ◽

In Vitro Binding ◽

Vitro Binding

During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar–phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.

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Transcription factor-like 5 is a potential DNA/RNA-binding protein essential for maintaining male fertility in mice. Running title: Tcfl5 is haploinsufficient in vivo

10.1101/2021.06.17.448807 ◽

2021 ◽

Author(s):

Weiya Xu ◽

Yiyun Zhang ◽

Dongdong Qin ◽

Yiqian Gui ◽

Shu Wang ◽

...

Keyword(s):

Transcription Factor ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fragile X ◽

Specific Protein ◽

Dual Function ◽

Specific Cell ◽

Chip Sequencing ◽

Dna And Rna

Tissue-specific transcription factors often play key roles in the development of specific cell lineages. Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, although the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/-) were infertile. We did, however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally regulated a set of genes participating in male germ cell development, which we uncovered via RNA-sequencing and TCFL5 ChIP-sequencing. We also found that TCFL5 interacted with RNA-binding proteins (RBPs) that regulated RNA processing, and further identified the fragile X mental retardation gene 1, autosomal homolog (FXR1, a known RBP) as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and hypothesize that TCFL5 may be a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.

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Detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct, long-read sequencing

10.1101/2021.11.03.467190 ◽

2021 ◽

Author(s):

Sepideh Tavakoli ◽

Mohammad Nabizadehmashhadtoroghi ◽

Amr Makhamreh ◽

Howard Gamper ◽

Neda Rezapour ◽

...

Keyword(s):

Single Molecule ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Parameters ◽

Type I ◽

High Confidence ◽

Rna Molecules ◽

Base Calling ◽

Long Read

Enzyme-mediated chemical modifications to mRNAs have the potential to fine-tune gene expression in response to environmental stimuli. Notably, pseudouridine-modified mRNAs are more resistant to RNase-mediated degradation, more responsive to cellular stress, and have the potential to modulate immunogenicity and enhance translation in vivo. However, the precise biological functions of pseudouridine modification on mRNAs remain unclear due to the lack of sensitive and accurate tools for mapping. We developed a semi-quantitative method for mapping pseudouridylated sites with high confidence directly on mammalian mRNA transcripts via direct RNA, long-read nanopore sequencing. By analysis of a modification-free transcriptome, we demonstrate that the depth of coverage and intrinsic errors associated with specific k-mer sequences are critical parameters for accurate base-calling. We adjust these parameters for high-confidence U-to-C base-calling errors that occur at pseudouridylated sites, which are benchmarked against sites that were identified previously by biochemical methods. We also uncovered new pseudouridylated sites, many of which fall on genes that encode RNA binding proteins and on uridine-rich k-mers. Sites identified by U-to-C base calling error were verified using 1000mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating that 1. the U-to-C base-calling error occurs at the site of pseudouridylation, and 2. the basecalling error is systematically under-calling the pseudouridylated sites. High-occupancy sites with >40% U-to-C basecalling error are classified as sites of hyper modification type I, whereas genes with more than one site of pseudouridylation are classified as having type II hyper modification which is confirmed by single-molecule analysis. We report the discovery of mRNAs with up to 7 unique sites of pseudouridine modification. Here we establish an innovative pipeline for direct identification, quantification, and detection of pseudouridine modifications and type I/II hypermodifications on native RNA molecules using long-read sequencing without resorting to RNA amplification, chemical reactions on RNA, enzyme-based replication, or DNA sequencing steps.

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RNA-mediated gene regulation is less evolvable than transcriptional regulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719138115 ◽

2018 ◽

Vol 115 (15) ◽

pp. E3481-E3490 ◽

Cited By ~ 11

Author(s):

Joshua L. Payne ◽

Fahad Khalid ◽

Andreas Wagner

Keyword(s):

Gene Regulation ◽

Transcription Factor ◽

Transcriptional Regulation ◽

Transcription Factors ◽

Dna Sequences ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Protein ◽

Regulatory Region

Much of gene regulation is carried out by proteins that bind DNA or RNA molecules at specific sequences. One class of such proteins is transcription factors, which bind short DNA sequences to regulate transcription. Another class is RNA binding proteins, which bind short RNA sequences to regulate RNA maturation, transport, and stability. Here, we study the robustness and evolvability of these regulatory mechanisms. To this end, we use experimental binding data from 172 human and fruit fly transcription factors and RNA binding proteins as well as human polymorphism data to study the evolution of binding sites in vivo. We find little difference between the robustness of regulatory protein–RNA interactions and transcription factor–DNA interactions to DNA mutations. In contrast, we find that RNA-mediated regulation is less evolvable than transcriptional regulation, because mutations are less likely to create interactions of an RNA molecule with a new RNA binding protein than they are to create interactions of a gene regulatory region with a new transcription factor. Our observations are consistent with the high level of conservation observed for interactions between RNA binding proteins and their target molecules as well as the evolutionary plasticity of regulatory regions bound by transcription factors. They may help explain why transcriptional regulation is implicated in many more evolutionary adaptations and innovations than RNA-mediated gene regulation.

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In vivo discovery of RNA proximal proteins in human cells via proximity-dependent biotinylation

10.1101/2020.02.28.970442 ◽

2020 ◽

Cited By ~ 1

Author(s):

Xianzhi Lin ◽

Marcos A. S. Fonseca ◽

Rosario I. Corona ◽

Kate Lawrenson

Keyword(s):

Gene Expression ◽

Ascorbate Peroxidase ◽

Genome Organization ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Components ◽

Rna Molecules ◽

Related Proteins ◽

Target Rna

AbstractRNA molecules function as messengers or noncoding adaptor molecules, structural components, and regulators of genome organization and gene expression. Their roles and regulation are mediated by other molecules they interact with, especially RNA binding proteins (RBPs). Here we report RNA proximity labeling (RPL), an RNA-centric method based on fusion of an endonuclease-deficient Type VI CRISPR-Cas protein (dCas13b) and engineered ascorbate peroxidase (APEX2) to discover in vivo target RNA proximal proteins (RPPs) through proximity-based biotinylation. U1 RPPs enriched by proximity-based biotinylation included both U1 snRNA canonical and noncanonical functions-related proteins. In addition, profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs for poly(A) tails and also provided novel evidence for poly(A)+ RNA 5’-3’ proximity and expanded subcellular localizations. Our results suggest that RPL is a rapid approach for identifying both interacting and neighboring proteins associated with target RNA molecules in their native cellular contexts.

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Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

Biochemistry and Cell Biology ◽

10.1139/o05-062 ◽

2005 ◽

Vol 83 (4) ◽

pp. 535-547 ◽

Cited By ~ 7

Author(s):

Gareth N Corry ◽

D Alan Underhill

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activation ◽

Current Knowledge ◽

Nuclear Architecture ◽

Subnuclear Localization ◽

Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

Methods and Protocols ◽

10.3390/mps4010022 ◽

2021 ◽

Vol 4 (1) ◽

pp. 22

Author(s):

Mrinmoyee Majumder ◽

Viswanathan Palanisamy

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Patterns ◽

Control Of Gene Expression ◽

Regulatory Pathways ◽

Advantages And Disadvantages ◽

Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

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RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

Non-Coding RNA ◽

10.3390/ncrna7010011 ◽

2021 ◽

Vol 7 (1) ◽

pp. 11 ◽

Cited By ~ 1

Author(s):

André P. Gerber

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Regulatory Networks ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Cell Protein ◽

Transcriptional Regulatory Networks ◽

Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

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