cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.26318171.v1
title: Enhanced binding of guanylated poly(A) RNA by the LaM domain of LARP1

description: La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3‘,3’-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation.
DOI: 10.6084/m9.figshare.27266911
title: Identification and functional characterization of lncRNAs involved in human monocyte-to-macrophage differentiation

description: Although long noncoding RNAs (lncRNAs) constitute the majority of the human transcriptome, the functional roles of most remain elusive. While protein-coding genes in macrophage biology have been extensively studied, the contribution of lncRNAs in this context is poorly understood. Given the vast number of lncRNAs (&gt;20,000), identifying candidates for functional characterization poses a significant challenge. Here, we present two complementary approaches to pinpoint and investigate lncRNAs involved in monocyte-to-macrophage differentiation: RNA-seq for functional inference and a high-throughput functional screen. These strategies enabled us to identify four lncRNA regulators of monocyte differentiation: <i>lincRNA-JADE1</i>, <i>lincRNA-ANXA3</i>, <i>GATA2-AS1</i>, and <i>PPP2R5C-AS1</i>. Preliminary insights suggest these lncRNAs may act in <i>cis</i> through neighbouring protein-coding genes, although their precise mechanisms remain to be elucidated. We further discuss the strengths and weaknesses of these methodologies, along with validation pipelines crucial for establishing lncRNA functionality.
DOI: 10.6084/m9.figshare.27266911.v1
title: Identification and functional characterization of lncRNAs involved in human monocyte-to-macrophage differentiation

description: Although long noncoding RNAs (lncRNAs) constitute the majority of the human transcriptome, the functional roles of most remain elusive. While protein-coding genes in macrophage biology have been extensively studied, the contribution of lncRNAs in this context is poorly understood. Given the vast number of lncRNAs (&gt;20,000), identifying candidates for functional characterization poses a significant challenge. Here, we present two complementary approaches to pinpoint and investigate lncRNAs involved in monocyte-to-macrophage differentiation: RNA-seq for functional inference and a high-throughput functional screen. These strategies enabled us to identify four lncRNA regulators of monocyte differentiation: <i>lincRNA-JADE1</i>, <i>lincRNA-ANXA3</i>, <i>GATA2-AS1</i>, and <i>PPP2R5C-AS1</i>. Preliminary insights suggest these lncRNAs may act in <i>cis</i> through neighbouring protein-coding genes, although their precise mechanisms remain to be elucidated. We further discuss the strengths and weaknesses of these methodologies, along with validation pipelines crucial for establishing lncRNA functionality.
DOI: 10.6084/m9.figshare.25231796
title: RNA-dependent proteome solubility maintenance in <i>Escherichia coli</i> lysates analysed by quantitative mass spectrometry: Proteomic characterization in terms of isoelectric point, structural disorder, functional hub, and chaperone network

description: Protein aggregation, a consequence of misfolding and impaired proteostasis, can lead to cellular malfunctions such as various proteinopathies. The mechanisms protecting proteins from aggregation in complex cellular environments have long been investigated, often from a protein-centric viewpoint. However, our study provides insights into a crucial, yet overlooked actor: RNA. We found that depleting RNAs from <i>Escherichia coli</i> lysates induces global protein aggregation. Our quantitative mass spectrometry analysis identified over 900 statistically significant proteins from the <i>Escherichia coli</i> proteome whose solubility depends on RNAs. Proteome-wide characterization showed that the RNA dependency is particularly enriched among acidic proteins, intrinsically disordered proteins, and structural hub proteins. Moreover, we observed distinct differences in RNA-binding mode and Gene Ontology categories between RNA-dependent acidic and basic proteins. Notably, the solubility of key molecular chaperones [Trigger factor, DnaJ, and GroES] is largely dependent on RNAs, suggesting a yet-to-be-explored hierarchical relationship between RNA-based chaperone (termed as chaperna) and protein-based chaperones, both of which constitute the whole chaperone network. These findings provide new insights into the RNA-centric role in maintaining healthy proteome solubility <i>in vivo</i>, where proteins associate with a variety of RNAs, either stably or transiently.
DOI: 10.6084/m9.figshare.25231796.v1
title: RNA-dependent proteome solubility maintenance in <i>Escherichia coli</i> lysates analysed by quantitative mass spectrometry: Proteomic characterization in terms of isoelectric point, structural disorder, functional hub, and chaperone network

description: Protein aggregation, a consequence of misfolding and impaired proteostasis, can lead to cellular malfunctions such as various proteinopathies. The mechanisms protecting proteins from aggregation in complex cellular environments have long been investigated, often from a protein-centric viewpoint. However, our study provides insights into a crucial, yet overlooked actor: RNA. We found that depleting RNAs from <i>Escherichia coli</i> lysates induces global protein aggregation. Our quantitative mass spectrometry analysis identified over 900 statistically significant proteins from the <i>Escherichia coli</i> proteome whose solubility depends on RNAs. Proteome-wide characterization showed that the RNA dependency is particularly enriched among acidic proteins, intrinsically disordered proteins, and structural hub proteins. Moreover, we observed distinct differences in RNA-binding mode and Gene Ontology categories between RNA-dependent acidic and basic proteins. Notably, the solubility of key molecular chaperones [Trigger factor, DnaJ, and GroES] is largely dependent on RNAs, suggesting a yet-to-be-explored hierarchical relationship between RNA-based chaperone (termed as chaperna) and protein-based chaperones, both of which constitute the whole chaperone network. These findings provide new insights into the RNA-centric role in maintaining healthy proteome solubility <i>in vivo</i>, where proteins associate with a variety of RNAs, either stably or transiently.
DOI: 10.6084/m9.figshare.26163224
title: TBP facilitates RNA Polymerase I transcription following mitosis

description: The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.
DOI: 10.6084/m9.figshare.26163224.v1
title: TBP facilitates RNA Polymerase I transcription following mitosis

description: The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.
DOI: 10.21233/rp4e-x927
title: TangbeiB testate amoebae surface sample dataset

description: Raw data for the TangbeiB testate amoebae surface sample dataset submitted to the Neotoma Paleoecology Database. Data is available through the landing page in JSON format. The landing page referenced by the DOI also contains links to publications and a map-based viewer for the dataset. The Neotoma Paleoecology Database maintains a homepage at https://www.neotomadb.org.
DOI: 10.6084/m9.figshare.24849248
title: Multiple Oligo assisted RNA Pulldown via Hybridization followed by Mass Spectrometry (MORPH-MS) for exploring the RNA-Protein interactions

description: Understanding RNA-protein interactions is crucial for deciphering the cellular functions and molecular mechanisms of regulatory RNAs. Consequently, there is a constant need to develop innovative and cost-effective methods to uncover such interactions. We developed a simple and cost-effective technique called Multiple Oligo assisted RNA Pulldown via Hybridization (MORPH) to identify proteins interacting with a specific RNA. MORPH employs a tiling array of antisense oligos (ASOs) to efficiently capture the RNA of interest along with proteins associated with it. Unlike existing techniques that rely on multiple individually biotinylated oligos spanning the entire RNA length, MORPH stands out by utilizing a single biotinylated oligo to capture all the ASOs. To evaluate MORPH’s efficacy, we applied this technique combined with mass spectrometry to identify proteins interacting with lncRNA NEAT1, which has previously been studied using various methods. Our results demonstrate that despite being a simple and inexpensive procedure, MORPH performs on par with existing methods. <b>Abbreviations</b>: ASO, Antisense oligo; lncRNA, long non-coding RNA; MORPH, Multiple Oligo assisted RNA Pulldown via Hybridization
DOI: 10.6084/m9.figshare.24849248.v1
title: Multiple Oligo assisted RNA Pulldown via Hybridization followed by Mass Spectrometry (MORPH-MS) for exploring the RNA-Protein interactions

description: Understanding RNA-protein interactions is crucial for deciphering the cellular functions and molecular mechanisms of regulatory RNAs. Consequently, there is a constant need to develop innovative and cost-effective methods to uncover such interactions. We developed a simple and cost-effective technique called Multiple Oligo assisted RNA Pulldown via Hybridization (MORPH) to identify proteins interacting with a specific RNA. MORPH employs a tiling array of antisense oligos (ASOs) to efficiently capture the RNA of interest along with proteins associated with it. Unlike existing techniques that rely on multiple individually biotinylated oligos spanning the entire RNA length, MORPH stands out by utilizing a single biotinylated oligo to capture all the ASOs. To evaluate MORPH’s efficacy, we applied this technique combined with mass spectrometry to identify proteins interacting with lncRNA NEAT1, which has previously been studied using various methods. Our results demonstrate that despite being a simple and inexpensive procedure, MORPH performs on par with existing methods. <b>Abbreviations</b>: ASO, Antisense oligo; lncRNA, long non-coding RNA; MORPH, Multiple Oligo assisted RNA Pulldown via Hybridization
DOI: 10.6084/m9.figshare.25132205
title: MiRNAs differentially expressed in vegetative and reproductive organs of <i>Marchantia polymorpha</i> – insights into their expression pattern, gene structures and function

description: MicroRNAs regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of <i>Marchantia polymorpha</i> makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that the differential expression of conserved miRNAs among land plants and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning.
DOI: 10.6084/m9.figshare.25132205.v1
title: MiRNAs differentially expressed in vegetative and reproductive organs of <i>Marchantia polymorpha</i> – insights into their expression pattern, gene structures and function

description: MicroRNAs regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of <i>Marchantia polymorpha</i> makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that the differential expression of conserved miRNAs among land plants and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning.
DOI: 10.6084/m9.figshare.25530825
title: Global and single-nucleotide resolution detection of 7-methylguanosine in RNA

description: RNA modifications, including <i>N</i>-7-methylguanosine (m<sup>7</sup>G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m<sup>7</sup>G modifications is of paramount significance, given recent associations between altered m<sup>7</sup>G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m<sup>7</sup>G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m<sup>7</sup>G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m<sup>7</sup>G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH<sub>4</sub>, neutral pH and the addition of 7-methylguanosine monophosphate (m<sup>7</sup>GMP) during the reducing reaction. Notably, compared to NaBH<sub>4</sub>-based methods, Bo-Seq enhances the m<sup>7</sup>G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m<sup>7</sup>G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m<sup>7</sup>G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m<sup>7</sup>G in gene expression and its implications in cancer biology.
DOI: 10.6084/m9.figshare.25530825.v1
title: Global and single-nucleotide resolution detection of 7-methylguanosine in RNA

description: RNA modifications, including <i>N</i>-7-methylguanosine (m<sup>7</sup>G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m<sup>7</sup>G modifications is of paramount significance, given recent associations between altered m<sup>7</sup>G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m<sup>7</sup>G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m<sup>7</sup>G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m<sup>7</sup>G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH<sub>4</sub>, neutral pH and the addition of 7-methylguanosine monophosphate (m<sup>7</sup>GMP) during the reducing reaction. Notably, compared to NaBH<sub>4</sub>-based methods, Bo-Seq enhances the m<sup>7</sup>G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m<sup>7</sup>G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m<sup>7</sup>G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m<sup>7</sup>G in gene expression and its implications in cancer biology.
DOI: 10.6084/m9.figshare.26144036.v1
title: Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre

description: Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on <i>Escherichia coli</i> growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the ‘critical region’ of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. <i>In vitro</i> fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis <i>in vivo</i>, as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg<sup>2+</sup>. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications <i>per se</i>, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.