Fluorescent Detection of Nascent Transcripts and RNA-binding Proteins in Cell Nuclei

The hnRNPs (heterogeneous nuclear ribonucleoproteins) are RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing and translational regulation. Although they share some general characteristics, they vary greatly in terms of their domain composition and functional properties. Although the traditional grouping of the hnRNPs as a collection of proteins provided a practical framework, which has guided much of the research on them, this approach is becoming increasingly incompatible with current knowledge about their structural and functional divergence. Hence, we review the current literature to examine hnRNP diversity, and discuss how this impacts upon approaches to the classification of RNA-binding proteins in general.

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Periphilin self-association underpins epigenetic silencing by the HUSH complex

10.1101/2019.12.18.881300 ◽

2019 ◽

Cited By ~ 2

Author(s):

Daniil M. Prigozhin ◽

Anna Albecka ◽

Christopher H. Douse ◽

Iva A. Tchasovnikarova ◽

Richard T. Timms ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Low Complexity ◽

Core Complex ◽

Terminal Domain ◽

Genome Wide ◽

Repressive Mark ◽

Self Association ◽

Nascent Transcripts

AbstractTranscription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin forms α-helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts.

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Insight Into Spinocerebellar Ataxia Type 31 (SCA31) From Drosophila Model

Frontiers in Neuroscience ◽

10.3389/fnins.2021.648133 ◽

2021 ◽

Vol 15 ◽

Author(s):

Taro Ishiguro ◽

Yoshitaka Nagai ◽

Kinya Ishikawa

Keyword(s):

Purkinje Cells ◽

Spinocerebellar Ataxia ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Spinocerebellar Ataxia Type ◽

Cell Nuclei ◽

Rna Foci ◽

Ppr Proteins ◽

Rna Chaperones

Spinocerebellar ataxia type 31 (SCA31) is a progressive neurodegenerative disease characterized by degeneration of Purkinje cells in the cerebellum. Its genetic cause is a 2.5- to 3.8-kb-long complex pentanucleotide repeat insertion containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n located in an intron shared by two different genes: brain expressed associated with NEDD4-1 (BEAN1) and thymidine kinase 2 (TK2). Among these repeat sequences, (TGGAA)n repeat was the only sequence segregating with SCA31, which strongly suggests its pathogenicity. In SCA31 patient brains, the mutant BEAN1 transcript containing expanded UGGAA repeats (UGGAAexp) was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. In addition, the deposition of pentapeptide repeat (PPR) proteins, poly(Trp-Asn-Gly-Met-Glu), translated from UGGAAexp RNA, was detected in the cytoplasm of Purkinje cells. To uncover the pathogenesis of UGGAAexp in SCA31, we generated Drosophila models of SCA31 expressing UGGAAexp RNA. The toxicity of UGGAAexp depended on its length and expression level, which was accompanied by the accumulation of RNA foci and translation of repeat-associated PPR proteins in Drosophila, consistent with the observation in SCA31 patient brains. We also revealed that TDP-43, FUS, and hnRNPA2B1, motor neuron disease–linked RNA-binding proteins bound to UGGAAexp RNA, act as RNA chaperones to regulate the formation of RNA foci and repeat-associated translation. Further research on the role of RNA-binding proteins as RNA chaperones may also provide a novel therapeutic strategy for other microsatellite repeat expansion diseases besides SCA31.

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Molecular Mechanisms and Future Therapeutics for Spinocerebellar Ataxia Type 31 (SCA31)

Neurotherapeutics ◽

10.1007/s13311-019-00804-6 ◽

2019 ◽

Vol 16 (4) ◽

pp. 1106-1114 ◽

Cited By ~ 5

Author(s):

Kinya Ishikawa ◽

Yoshitaka Nagai

Keyword(s):

Spinocerebellar Ataxia ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Spinocerebellar Ataxia Type ◽

Cell Nuclei ◽

Rna Foci ◽

Progressive Cerebellar Ataxia ◽

Cardinal Symptom

AbstractSpinocerebellar ataxia type 31 (SCA31) is one of the autosomal-dominant neurodegenerative disorders that shows progressive cerebellar ataxia as a cardinal symptom. This disease is caused by a 2.5- to 3.8-kb-long complex pentanucleotide repeat containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n in an intron of the gene called BEAN1 (brain expressed, associated with Nedd4). By comparing various pentanucleotide repeats in this particular locus among control Japanese and Caucasian populations, it was found that (TGGAA)n was the only sequence segregating with SCA31, strongly suggesting the pathogenicity of (TGGAA)n. The complex repeat also lies in an intron of another gene, TK2 (thymidine kinase 2), which is transcribed in the opposite direction, indicating that the complex repeat is bi-directionally transcribed as noncoding repeats. In SCA31 human brains, (UGGAA)n, the BEAN1 transcript of SCA31 mutation was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. Subsequent RNA pulldown analysis disclosed that (UGGAA)n binds to RNA-binding proteins TDP-43, FUS, and hnRNP A2/B1. In fact, TDP-43 was found to co-localize with RNA foci in human SCA31 Purkinje cells. To dissect the pathogenesis of (UGGAA)n in SCA31, we generated transgenic fly models of SCA31 by overexpressing SCA31 complex pentanucleotide repeats in Drosophila. We found that the toxicity of (UGGAA)n is length- and expression level–dependent, and it was dampened by co-expressing TDP-43, FUS, and hnRNP A2/B1. Further investigation revealed that TDP-43 ameliorates (UGGAA)n toxicity by directly fixing the abnormal structure of (UGGAA)n. This led us to propose that TDP-43 acts as an RNA chaperone against toxic (UGGAA)n. Further research on the role of RNA-binding proteins as RNA chaperones may provide a novel therapeutic strategy for SCA31.

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The RNA-binding proteins CELF1 and ELAVL1 cooperatively control RNA isoform production

10.1101/373704 ◽

2018 ◽

Author(s):

Géraldine David ◽

David Reboutier ◽

Stéphane Deschamps ◽

Agnès Méreau ◽

William Taylor ◽

...

Keyword(s):

Alternative Splicing ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Limited Information ◽

Cell Nuclei ◽

Rt Pcr ◽

Human Pathology ◽

Mrna Isoform ◽

Additive Control

ABSTRACTELAVL1 and CELF1 are RNA-binding proteins that are involved in alternative splicing control. To explore their functional relationship, we looked for mRNAs that are differentially spliced following the depletion of CELF1, ELAVL1, or both. We found that these proteins control the usage of their target exons in the same direction. Double depletion has a greater effect than individual depletions, showing that CELF1 and ELAVL1 exert additive control. To confirm these results, we carried out RT-PCR on the alternative cassette exons of several mRNAs, including CD44, WNK1, PHACTR2, MICAL3, SPTBN1, and PPP3CB. Using FRET, we found that CELF1 and ELAVL1 directly interact in cell nuclei. We demonstrated that the combined levels of CELF1 and ELAVL1 are a valuable biomarker in several cancers, even when their individual levels may yield very limited information. CD44 alternative splicing probably accounts in part for the effects of CELF1 and ELAVL1 levels on patient survival. These data point to strong functional interactions between CELF1 and ELAVL1 in the control of mRNA isoform production, resulting in significant impacts on human pathology.

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