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Widespread transcriptional readthrough caused by Nab2 depletion leads to chimeric transcripts with retained introns

ABSTRACT: Nascent RNA sequencing has recently revealed that pre-mRNA splicing can occur shortly after the intron emerges from RNA polymerase II (Pol II). Differences in co-transcriptional splicing profiles suggest regulation by cis- and/or trans-acting factors. Here we used Single Molecule Intron Tracking (SMIT) in budding yeast to identify a cohort of regulators by machine learning. One candidate, Nab2, displayed reduced co-transcriptional splicing of some pre-mRNAs when depleted. Unexpectedly, these splicing defects were attributable to readthrough transcription, which was revealed by long read sequencing of nascent RNA; individual readthrough transcripts induced by Nab2 depletion sometimes spanned multiple genes. Thus, Nab2 regulation of splicing was indirect. Moreover, unspliced transcripts displayed downstream readthrough in both control and Nab2-depleted cells, highlighting the coupling between splicing and 3′ end formation. We conclude that Nab2 is required for proper 3′ end processing, which ensures both transcription termination as well as proper splicing of downstream genes.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE156133 | GEO | 2020/10/27

REPOSITORIES: GEO

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The Nab2 RNA binding protein promotes sex-specific splicing of Sex lethal in Drosophila neuronal tissue

Project description:The Drosophila polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls axon projection, locomotion, and memory. Here we define an unexpectedly specific role for Nab2 in regulating splicing of ~150 exons/introns in the head transcriptome and link the most prominent of these, female retention of a male-specific exon in the sex determination factor Sex-lethal (Sxl), to a role in m6A-dependent mRNA splicing. Genetic evidence indicates that aberrant Sxl splicing underlies multiple phenotypes in Nab2 mutant females. At a molecular level, Nab2 associates with Sxl pre-mRNA and ensures proper female-specific splicing by preventing m6A hypermethylation by Mettl3 methyltransferase. Consistent with these results, reducing Mettl3 expression rescues developmental, behavioral and neuroanatomical phenotypes in Nab2 mutants. Overall these data identify Nab2 as a required regulator of m6A-regulated Sxl splicing and imply a broader link between Nab2 and Mettl3-regulated brain RNAs.

2020-12-02 | GSE162531 | GEO

Widespread transcriptional readthrough caused by Nab2 depletion leads to chimeric transcripts with retained introns

Project description:Widespread transcriptional readthrough caused by Nab2 depletion leads to chimeric transcripts with retained introns

| PRJNA656824 | ENA

Expression profile of the “heat-inducible N-degron” of Nab2 (nab2-td)

Project description:Gene expression in eukaryotes is an essential process that includes transcription, pre-RNA processing and RNA export. All these steps are coupled and normally, any failure in one step affects the other steps and could cause nuclear mRNA retention. One important player in this interface is the poly(A)-RNA binding protein Nab2, which regulates the poly(A)-tail length of mRNAs protecting their 3’-ends from a second round of polyadenylation and facilitating their nucleo-cytoplasmic export. Interestingly, here we show that Nab2 has additional roles in mRNA transcription elongation, tRNA metabolism and rRNA export. We use Genome-wide analysis of expression of a conditional degron nab2 mutant that allows the depletion of the protein in 15 minutes, to demosntrate that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is not the result of a secondary effect. Our results identify primary targets of nab2 revealing novel functions for Nab2 in transcription and metabolism of most types of RNAs, not only poly(A) mRNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, being a central player in the general and coordinated control of gene expression from transcription elongation to translation initiation.

2011-12-01 | GSE19302 | GEO

Expression profile of nab2-1 mutant

Project description:Gene expression in eukaryotes is an essential process that includes transcription, pre-RNA processing and RNA export. All these steps are coupled and normally, any failure in one step affects the other steps and could cause nuclear mRNA retention. One important player in this interface is the poly(A)-RNA binding protein Nab2, which regulates the poly(A)-tail length of mRNAs protecting their 3’-ends from a second round of polyadenylation and facilitating their nucleo-cytoplasmic export. Interestingly, here we show that Nab2 has additional roles in mRNA transcription elongation, tRNA metabolism and rRNA export. We use Genome-wide analysis of expression of nab2-1 mutant to demosntrate that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is not the result of a secondary effect. Our results identify primary targets of nab2 revealing novel functions for Nab2 in transcription and metabolism of most types of RNAs, not only poly(A) mRNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, being a central player in the general and coordinated control of gene expression from transcription elongation to translation initiation.

2011-12-01 | GSE26303 | GEO

Expression profile of nab2-1 mutant

Project description:Gene expression in eukaryotes is an essential process that includes transcription, pre-RNA processing and RNA export. All these steps are coupled and normally, any failure in one step affects the other steps and could cause nuclear mRNA retention. One important player in this interface is the poly(A)-RNA binding protein Nab2, which regulates the poly(A)-tail length of mRNAs protecting their 3M-bM-^@M-^Y-ends from a second round of polyadenylation and facilitating their nucleo-cytoplasmic export. Interestingly, here we show that Nab2 has additional roles in mRNA transcription elongation, tRNA metabolism and rRNA export. We use Genome-wide analysis of expression of nab2-1 mutant to demosntrate that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is not the result of a secondary effect. Our results identify primary targets of nab2 revealing novel functions for Nab2 in transcription and metabolism of most types of RNAs, not only poly(A) mRNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, being a central player in the general and coordinated control of gene expression from transcription elongation to translation initiation. Three repeats of the wild type and nab2-1 mutant

2011-12-01 | E-GEOD-26303 | biostudies-arrayexpress

The disease-associated proteins Drosophila Nab2 and Ataxin-2 interact with shared RNAs and coregulate neuronal morphology

Project description:Nab2 encodes a polyadenosine RNA-binding protein (RBP) with broad roles in post-transcriptional regulation, including in RNA export, poly(A) tail length control, and mRNA splicing, and loss of its human ortholog ZC3H14 gives rise to a form of autosomal recessive intellectual disability. Understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part, because no comprehensive identification of metazoan-Nab2-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships likely remain unidentified. Here we present evidence that Drosophila melanogaster Nab2 interacts with the RBP Ataxin-2 (Atx2), a neuronal translational regulator, and implicate these proteins in coordinate regulation of neuronal morphology and adult viability. We then present the first high-throughput identifications of RNAs associating with Nab2 and Atx2 in Drosophila brain neurons using an RNA immunoprecipitation-sequencing (RIP-Seq) approach. Critically, the RNA interactomes of each RBP overlap. The identities of shared associated transcripts (e.g. drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g. Arpc2, tea, respectively) promise insight into the neuronal functions of and interactions between each RBP. Significantly, we find Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with only a fraction of all polyadenylated RNAs. These Nab2-associated RNAs are overrepresented for internal A-rich motifs, suggesting such sequences may partially mediate Nab2 target selection. Taken together, these data demonstrate 1)Nab2 opposingly regulates neuronal morphology and shares associated neuronal RNAs with Atx2 and 2)Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.

2021-02-05 | GSE165677 | GEO

Expression profile of the M-bM-^@M-^\heat-inducible N-degronM-bM-^@M-^] of Nab2 (nab2-td)

Project description:Gene expression in eukaryotes is an essential process that includes transcription, pre-RNA processing and RNA export. All these steps are coupled and normally, any failure in one step affects the other steps and could cause nuclear mRNA retention. One important player in this interface is the poly(A)-RNA binding protein Nab2, which regulates the poly(A)-tail length of mRNAs protecting their 3M-bM-^@M-^Y-ends from a second round of polyadenylation and facilitating their nucleo-cytoplasmic export. Interestingly, here we show that Nab2 has additional roles in mRNA transcription elongation, tRNA metabolism and rRNA export. We use Genome-wide analysis of expression of a conditional degron nab2 mutant that allows the depletion of the protein in 15 minutes, to demosntrate that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is not the result of a secondary effect. Our results identify primary targets of nab2 revealing novel functions for Nab2 in transcription and metabolism of most types of RNAs, not only poly(A) mRNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, being a central player in the general and coordinated control of gene expression from transcription elongation to translation initiation. Two and three repeats of the wild type control and the nab2-td mutant respectively at time 0, 30 and 75 minutes after the degron induction.

2011-12-01 | E-GEOD-19302 | biostudies-arrayexpress

Transcription profiling by array of Nab2 depletion effect on yeast mRNA

Project description:Transcription profiling was performed on the time course of Nab2 protein depletion in THC-Nab2 strain at 0h, 7h, 12h in biological triplicates to access its affect on the composition and structure of mRNA in budding yeast. For the protein depletion experiment THC-NAB2 (THJ collection Y2484, THC-NAB2:KAN tTA:URA, Open Biosystems) strain was used, where expression of the NAB2 gene is under control of a tetracyclin-repressible promoter.

2013-01-11 | E-MTAB-981 | biostudies-arrayexpress

Glucose stress causes mRNA retention in nuclear Nab2 condensates

Project description:Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that upon glucose withdrawal the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and to control gene expression during stress.

2023-12-19 | GSE247953 | GEO

Nab2 Drosophila pupal brain LC-MSMS

Project description:We exploit the predictable time course of Drosophila brain development to perform a temporally coupled quantitative proteomic analysis of the pupal brain in Nab2 mutant or overexpression models, which reveals that Nab2 is required to regulate the abundance of a number of proteins with critical roles in Drosophila neurons. Pupal brains lacking Nab2 show dysregulation of proteins, such as Futsch, Turtle, Contactin, and Van Gogh, that typically function in brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycle, and other neurodevelopmental processes. Overall, these data define a role for Nab2 during neurodevelopment in regulating protein abundance for a subset of the brain proteome and provide a window into the potential functions of human ZC3H14 protein.

2022-02-17 | PXD022984 | Pride

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