Heidar J Koning, Jia Y Lai, Andrew C Marshall, Elke Stroeher, Gavin Monahan, Anuradha Pullakhandam, Gavin J Knott, Timothy M Ryan, Archa H Fox, Andrew Whitten, Mihwa Lee, Charles S Bond
{"title":"Structural plasticity of the coiled-coil interactions in human SFPQ.","authors":"Heidar J Koning, Jia Y Lai, Andrew C Marshall, Elke Stroeher, Gavin Monahan, Anuradha Pullakhandam, Gavin J Knott, Timothy M Ryan, Archa H Fox, Andrew Whitten, Mihwa Lee, Charles S Bond","doi":"10.1093/nar/gkae1198","DOIUrl":"https://doi.org/10.1093/nar/gkae1198","url":null,"abstract":"<p><p>The proteins SFPQ (splicing Factor Proline/Glutamine rich) and NONO (non-POU domain-containing octamer-binding protein) are mammalian members of the Drosophila Behaviour/Human Splicing (DBHS) protein family, which share 76% sequence identity in their conserved 320 amino acid DBHS domain. SFPQ and NONO are involved in all steps of post-transcriptional regulation and are primarily located in mammalian paraspeckles: liquid phase-separated, ribonucleoprotein sub-nuclear bodies templated by NEAT1 long non-coding RNA. A combination of structured and low-complexity regions provide polyvalent interaction interfaces that facilitate homo- and heterodimerisation, polymerisation, interactions with oligonucleotides, mRNA, long non-coding RNA, and liquid phase-separation, all of which have been implicated in cellular homeostasis and neurological diseases including neuroblastoma. The strength and competition of these interaction modes define the ability of DBHS proteins to dissociate from paraspeckles to fulfil functional roles throughout the nucleus or the cytoplasm. In this study, we define and dissect the coiled-coil interactions which promote the polymerisation of DBHS proteins, using a crystal structure of an SFPQ/NONO heterodimer which reveals a flexible coiled-coil interaction interface which differs from previous studies. We support this through extensive solution small-angle X-ray scattering experiments using a panel of SFPQ/NONO heterodimer variants which are capable of tetramerisation to varying extents. The QM mutant displayed a negligible amount of tetramerisation (quadruple loss of function coiled-coil mutant L535A/L539A/L546A/M549A), the Charged Single Alpha Helix (ΔCSAH) variant displayed a dimer-tetramer equilibrium interaction, and the disulfide-forming variant displayed constitutive tetramerisation (R542C which mimics the pathological Drosophila nonAdiss allele). We demonstrate that newly characterised coiled-coil interfaces play a role in the polymerisation of DBHS proteins in addition to the previously described canonical coiled-coil interface. The detail of these interactions provides insight into a process critical for the assembly of paraspeckles as well as the behaviour of SFPQ as a transcription factor, and general multipurpose auxiliary protein with functions essential to mammalian life. Our understanding of the coiled coil behaviour of SFPQ also enhances the explanatory power of mutations (often disease-associated) observed in the DBHS family, potentially allowing for the development of future medical options such as targeted gene therapy.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854860","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dongju An, Jihyun Kim, Byul Moon, Hyoungmin Kim, Hoa Nguyen, Sunghu Park, J Eugene Lee, Jung-Ae Kim, Jaehoon Kim
{"title":"PRMT1-mediated methylation regulates MLL2 stability and gene expression.","authors":"Dongju An, Jihyun Kim, Byul Moon, Hyoungmin Kim, Hoa Nguyen, Sunghu Park, J Eugene Lee, Jung-Ae Kim, Jaehoon Kim","doi":"10.1093/nar/gkae1227","DOIUrl":"https://doi.org/10.1093/nar/gkae1227","url":null,"abstract":"<p><p>The interplay between multiple transcription factors precisely regulates eukaryotic transcription. Here, we report that the protein methyltransferases, MLL2/KMT2B and PRMT1, interact directly and act collectively to regulate gene expression. PRMT1 binds to the N-terminal region of MLL2, considered an intrinsically disordered region, and methylates multiple arginine residues within its RGG/RG motifs. Notably, overexpression of PRMT1 decreased poly-ubiquitylation of MLL2, whereas mutations on methylation sites in MLL2 increased MLL2 poly-ubiquitylation, suggesting that PRMT1-mediated methylation stabilizes MLL2. MLL2 and PRMT1 cooperatively stimulated the expression of a chromosomal reporter gene in a PRMT1-mediated, MLL2-methylation-dependent manner. RNA-seq analysis found that MLL2 and PRMT1 jointly regulate the expression of genes involved in cell membrane and extracellular matrix functions, and depletion of either resulted in impaired cell migration and invasion. Our study provides evidence that PRMT1-mediated MLL2 methylation regulates MLL2 protein stability and the expression of their target genes.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Manuel Brenes-Álvarez, Halie R Ropp, Dimitrios Papagiannidis, Clement M Potel, Frank Stein, Ingeborg Scholz, Claudia Steglich, Mikhail M Savitski, Agustín Vioque, Alicia M Muro-Pastor, Wolfgang R Hess
{"title":"R-DeeP/TripepSVM identifies the RNA-binding OB-fold-like protein PatR as regulator of heterocyst patterning.","authors":"Manuel Brenes-Álvarez, Halie R Ropp, Dimitrios Papagiannidis, Clement M Potel, Frank Stein, Ingeborg Scholz, Claudia Steglich, Mikhail M Savitski, Agustín Vioque, Alicia M Muro-Pastor, Wolfgang R Hess","doi":"10.1093/nar/gkae1247","DOIUrl":"https://doi.org/10.1093/nar/gkae1247","url":null,"abstract":"<p><p>RNA-binding proteins (RBPs) are central components of gene regulatory networks. The differentiation of heterocysts in filamentous cyanobacteria is an example of cell differentiation in prokaryotes. Although multiple non-coding transcripts are involved in this process, no RBPs have been implicated thus far. Here we used quantitative mass spectrometry to analyze the differential fractionation of RNA-protein complexes after RNase treatment in density gradients yielding 333 RNA-associated proteins, while a bioinformatic prediction yielded 311 RBP candidates in Nostoc sp. PCC 7120. We validated in vivo the RNA-binding capacity of six RBP candidates. Some participate in essential physiological aspects, such as photosynthesis (Alr2890), thylakoid biogenesis (Vipp1) or heterocyst differentiation (PrpA, PatU3), but their association with RNA was unknown. Validated RBPs Asl3888 and Alr1700 were not previously characterized. Alr1700 is an RBP with two oligonucleotide/oligosaccharide-binding (OB)-fold-like domains that is differentially expressed in heterocysts and interacts with non-coding regulatory RNAs. Deletion of alr1700 led to complete deregulation of the cell differentiation process, a striking increase in the number of heterocyst-like cells, and was ultimately lethal in the absence of combined nitrogen. These observations characterize this RBP as a master regulator of the heterocyst patterning and differentiation process, leading us to rename Alr1700 to PatR.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854843","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Correction to \"Growth-regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes\".","authors":"","doi":"10.1093/nar/gkae1258","DOIUrl":"https://doi.org/10.1093/nar/gkae1258","url":null,"abstract":"","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864925","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
David M English, Samuel N Lee, Khadija A Sabat, India M Baker, Trong Khoa Pham, Mark O Collins, Shaun M Cowley
{"title":"Rapid degradation of histone deacetylase 1 (HDAC1) reveals essential roles in both gene repression and active transcription.","authors":"David M English, Samuel N Lee, Khadija A Sabat, India M Baker, Trong Khoa Pham, Mark O Collins, Shaun M Cowley","doi":"10.1093/nar/gkae1223","DOIUrl":"https://doi.org/10.1093/nar/gkae1223","url":null,"abstract":"<p><p>Histone Deacetylase 1 (HDAC1) removes acetyl groups from lysine residues on core histones, a critical step in regulating chromatin accessibility. Despite histone deacetylation being an apparently repressive activity, suppression of HDACs causes both up- and downregulation of gene expression. Here we exploited the degradation tag (dTAG) system to rapidly degrade HDAC1 in mouse embryonic stem cells (ESCs) lacking its paralog, HDAC2. The dTAG system allowed specific degradation and removal of HDAC1 in <1 h (100x faster than genetic knockouts). This rapid degradation caused increased histone acetylation in as little as 2 h, with H2BK5 and H2BK11 being the most sensitive. The majority of differentially expressed genes following 2 h of HDAC1 degradation were upregulated (275 genes up versus 15 down) with increased proportions of downregulated genes observed at 6 h (1153 up versus 443 down) and 24 h (1146 up versus 967 down), respectively. Upregulated genes showed increased H2BK5ac and H3K27ac around their transcriptional start site (TSS). In contrast, decreased acetylation and chromatin accessibility of super-enhancers was linked to the most strongly downregulated genes. These findings suggest a paradoxical role for HDAC1 in the maintenance of histone acetylation levels at critical enhancer regions required for the pluripotency-associated gene network.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142865034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Protein-free catalysis of DNA hydrolysis and self-integration by a ribozyme.","authors":"Deni Szokoli, Hannes Mutschler","doi":"10.1093/nar/gkae1224","DOIUrl":"https://doi.org/10.1093/nar/gkae1224","url":null,"abstract":"<p><p>Group II introns are ancient self-splicing ribozymes and retrotransposons. Though long speculated to have originated before translation, their dependence on intron-encoded proteins for splicing and mobility has cast doubt on this hypothesis. While some group II introns are known to retain part of their catalytic repertoire in the absence of protein cofactors, protein-free complete reverse splicing of a group II intron into a DNA target has never been demonstrated. Here, we demonstrate the complete independence of a group II intron from protein cofactors in all intron-catalyzed reactions. The ribozyme is capable of fully reverse splicing into single-stranded DNA targets in vitro, readily hydrolyzes DNA substrates and is even able to unwind and react with stably duplexed DNA. Our findings make a protein-free origin for group II introns plausible by expanding their known catalytic capabilities beyond what would be needed to survive the transition from RNA to DNA genomes. Furthermore, the intron's capacity to react with both single and double-stranded DNA in conjunction with its expanded sequence recognition may represent a promising starting point for the development of protein-free genomic editing tools.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Katarzyna M Soczek, Joshua C Cofsky, Owen T Tuck, Honglue Shi, Jennifer A Doudna
{"title":"CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.","authors":"Katarzyna M Soczek, Joshua C Cofsky, Owen T Tuck, Honglue Shi, Jennifer A Doudna","doi":"10.1093/nar/gkae1192","DOIUrl":"10.1093/nar/gkae1192","url":null,"abstract":"<p><p>RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Correction to 'Accelerated discovery and miniaturization of novel single-stranded cytidine deaminases'.","authors":"","doi":"10.1093/nar/gkae1287","DOIUrl":"https://doi.org/10.1093/nar/gkae1287","url":null,"abstract":"","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864926","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kate R Harding, Lucia M Malone, Natalie A P Kyte, Simon A Jackson, Leah M Smith, Peter C Fineran
{"title":"Genome-wide identification of bacterial genes contributing to nucleus-forming jumbo phage infection","authors":"Kate R Harding, Lucia M Malone, Natalie A P Kyte, Simon A Jackson, Leah M Smith, Peter C Fineran","doi":"10.1093/nar/gkae1194","DOIUrl":"https://doi.org/10.1093/nar/gkae1194","url":null,"abstract":"The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage ‘nucleus’ compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"59 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142848979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}