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1.
EMBO Rep ; 23(9): e55432, 2022 09 05.
Article in English | MEDLINE | ID: mdl-35856391

ABSTRACT

The P-TEFb complex promotes transcription elongation by releasing paused RNA polymerase II. P-TEFb itself is known to be inactivated through binding to the non-coding RNA 7SK but there is only limited information about mechanisms regulating their association. Here, we show that cells deficient in the RNA-binding protein hnRNP R, a known 7SK interactor, exhibit increased transcription due to phosphorylation of RNA polymerase II. Intriguingly, loss of hnRNP R promotes the release of P-TEFb from 7SK, accompanied by enhanced hnRNP A1 binding to 7SK. Additionally, we found that hnRNP R interacts with BRD4, and that hnRNP R depletion increases BRD4 binding to the P-TEFb component CDK9. Finally, CDK9 is stabilized upon loss of hnRNP R and its association with Cyclin K is enhanced. Together, our results indicate that hnRNP R negatively regulates transcription by modulating the activity and stability of the P-TEFb complex, exemplifying the multimodal regulation of P-TEFb by an RNA-binding protein.


Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins , Nuclear Proteins , Positive Transcriptional Elongation Factor B , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , HeLa Cells , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Positive Transcriptional Elongation Factor B/genetics , Positive Transcriptional Elongation Factor B/metabolism , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Long Noncoding , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic
2.
Nucleic Acids Res ; 49(21): 12284-12305, 2021 12 02.
Article in English | MEDLINE | ID: mdl-34850154

ABSTRACT

Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnprtm1a/tm1a) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnprtm1a/tm1a mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with γ-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.


Subject(s)
Chromatin/genetics , DNA Damage , DNA Repair/genetics , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Motor Neurons/metabolism , Y-Box-Binding Protein 1/genetics , Animals , Axons/metabolism , Cell Line , Cells, Cultured , Chromatin/metabolism , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Immunoblotting , Mice, Inbred C57BL , Mice, Knockout , Motor Neurons/cytology , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Y-Box-Binding Protein 1/metabolism
3.
J Cell Sci ; 134(22)2021 11 15.
Article in English | MEDLINE | ID: mdl-34668554

ABSTRACT

In neurons, the endoplasmic reticulum (ER) forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance; however, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high-resolution microscopy and live-cell imaging to investigate rapid movements of the ER and ribosomes in axons of cultured motoneurons after stimulation with brain-derived neurotrophic factor. Our results indicate that the ER extends into axonal growth cone filopodia, where its integrity and dynamic remodeling are regulated mainly by actin and the actin-based motor protein myosin VI (encoded by Myo6). Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with the ER in response to extracellular stimuli, which describes a novel function of axonal ER in dynamic regulation of local translation. This article has an associated First Person interview with Chunchu Deng, joint first author of the paper.


Subject(s)
Axons , Presynaptic Terminals , Endoplasmic Reticulum , Humans , Motor Neurons , Ribosomes
4.
Nat Commun ; 12(1): 1278, 2021 02 24.
Article in English | MEDLINE | ID: mdl-33627647

ABSTRACT

Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.


Subject(s)
RNA, Long Noncoding/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , SMN Complex Proteins/metabolism , Animals , Cells, Cultured , HEK293 Cells , HeLa Cells , Humans , Mice , Motor Neurons/metabolism , RNA, Long Noncoding/genetics , Ribonucleoproteins, Small Nuclear/genetics , SMN Complex Proteins/genetics , Tandem Mass Spectrometry , Transcription, Genetic/genetics
5.
Proc Natl Acad Sci U S A ; 115(12): E2859-E2868, 2018 03 20.
Article in English | MEDLINE | ID: mdl-29507242

ABSTRACT

Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance.


Subject(s)
Axons/physiology , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Motor Neurons/physiology , RNA, Small Nuclear/metabolism , 3' Untranslated Regions , Animals , Cell Nucleus/genetics , Cytosol/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Immunoprecipitation/methods , Mice , RNA, Messenger/metabolism , RNA, Small Nuclear/genetics , Transcriptome/genetics
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