TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes

Kawahara, Yukio; Mieda-Sato, Ai

doi:10.1073/pnas.1112427109

Cited by 361 publications

(392 citation statements)

References 32 publications

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“…The cellular levels of many miRNAs are highly regulated depending on developmental stage and tissue type (21,22). Several specific RNA-binding proteins, including Lin28A, KH-type splicing regulatory protein (KSRP), TAR DNA binding protein-43 (TDP43), and heteronuclear ribonucleoprotein A1 (hnRNP A1), have been shown to bind precursor miRNAs and to affect miRNA biogenesis (17,(23)(24)(25). However, most miRNAs and their precursor transcripts have not been analyzed for specific binding proteins that may regulate their processing and biological functions.…”

Section: Resultsmentioning

confidence: 99%

RNA–protein analysis using a conditional CRISPR nuclease

Lee

Haurwitz

Apffel

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.non-coding RNA | RNA processing | miRNA | mass spectrometry R NA molecules function together with specific binding proteins to regulate cellular pathways at the levels of transcription, posttranscriptional modification, and translation (1-4). For example, microRNAs (miRNA), siRNAs, and piwi-interacting RNAs (piRNAs) regulate more than 30% of mammalian gene expression (5). Small nucleolar RNAs (snoRNAs) govern the sites and efficiencies of RNA chemical modifications in cells (6), whereas long noncoding RNAs (lncRNAs), such as HOTAIR and MALAT1, have been implicated in chromatin remodeling, transcriptional activation, and tumorigenesis (7,8). UTRs of mRNA transcripts are also known to interact with regulatory proteins to control their expression level as well as their stability. Understanding how these RNAs function in cells and how they may be manipulated for therapeutic purposes is an important goal that spans many areas of biology.Although new ncRNAs are being discovered at a rapid pace, determination of their biochemical activities is often slow. A major barrier to such functional analysis is the current difficulty of identifying RNA-binding partners that associate with specific transcripts and participate in their biological behavior. Despite the development of various RNA affinity purification methods, the expense and/or technical challenges associated with each approach has precluded its use by nonspecialists or in high-throughput discovery experiments. Current strategies for identifying RNA-binding proteins that associate with specific transcripts involve the use of affinity tags, including biotin, aptamers, and particular proteinbinding sequences (9-13). In each case, however, the modest affinity or specificity of tag recognition and the difficulty of selective elution complicate sample analysis. A strategy that will allow simple and rapid identification of proteins that associate selectively with particu...

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Section: Resultsmentioning

confidence: 99%

RNA–protein analysis using a conditional CRISPR nuclease

Lee

Haurwitz

Apffel

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…3 The presence of an intermediate state in the RRM unfolding pathways of TDP-43 and FUS/TLS suggests that these RRMs may serve to stabilize the other RNA-binding domain and sequester the NES within the hydrophobic core to control cytoplasmic shuttling or to prevent aggregation. RRM2 must partially unfold to expose the NES for recognition by exportin (76), which mediates the transport to the cytoplasm. This hypothesis suggests that cleavage within RRM2, loss of RRM1, or external stressors would increase the population of the intermediate state and disrupt the normal cellular distribution.…”

Section: Discussionmentioning

confidence: 99%

Folding of the RNA Recognition Motif (RRM) Domains of the Amyotrophic Lateral Sclerosis (ALS)-linked Protein TDP-43 Reveals an Intermediate State

Mackness

Tran

McClain

et al. 2014

Journal of Biological Chemistry

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“…To generate the expression construct for full-length TDP-43 tagged with SNAP at the N terminus and HaloTag at the C terminus (pSNAP-TDP-43(WT)-HTC), the coding region of TDP-43 was first amplified using the FALG-TDP-43 expression vector as the template 41 and inserted into the pFC14K vector (Promega) using the Sgf1 and EcoICR1 restriction sites to generate pFC14K-TDP-43-HTC. Next the TDP-43-HTC region was amplified and inserted into the pSNAP-tag(m) vector (NEB) using the Sbf1 and Not1 restriction sites.…”

Section: Methodsmentioning

confidence: 99%

The cleavage pattern of TDP-43 determines its rate of clearance and cytotoxicity

et al. 2015

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TAR DNA-binding protein of 43 kDa (TDP-43) and its C-terminal fragment of 25 kDa (CTF25) play critical roles in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although the overexpression of TDP-43 in cultured cells and animals results in the production of CTF25, the cleavage site that generates CTF25 and biological significance of the cleavage remain undetermined. Here we identify Asp174 as a cleavage site for CTF25. TDP-43 is cleaved initially after Asp174, which activates caspase-3/7 to accelerate TDP-43 fragmentation. Consequently, blockage of this cleavage results in a severe delay in TDP-43 clearance and prolonged necrotic cell death. We further show that the endoplasmic reticulum membrane-bound caspase-4 is the enzyme responsible for the cleavage after Asp174 and inhibition of caspase-4 activity slows TDP-43 fragmentation and reduces cell viability. These findings suggest that caspase-4-mediated cleavage after Asp174 is an initiator of TDP-43 clearance, which is required to avoid cell death induced by overexpressed TDP-43. A myotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases and is characterized by muscle weakness due to the degeneration of both upper and lower motor neurons. Although the pathogenic mechanism of ALS remains unresolved, the RNA-binding protein TDP-43 has been shown to accumulate in cytoplasmic inclusions in the affected regions of the spinal cord and brain in patients with ALS and frontotemporal lobar degeneration (FTLD), respectively 1,2 . Subsequently, mutations linked with ALS and FTLD have been identified in the gene that encodes TDP-43, which has confirmed the significance of TDP-43 in the pathogenesis of these diseases 3,4 . Most of these mutations occur within or near the carboxyl-terminal (C-terminal) glycine-rich domain (GRD) of TDP-43, except for the D169G point mutation that resides in RNA recognition motif 1 (refs 3,4). TDP-43 is normally localized predominantly in the nucleus. However, in ALS and FTLD patients with TDP-43-positive cytoplasmic inclusions, the protein is abnormally phosphorylated, ubiquitinated and truncated. This results in the accumulation of an B25-kDa C-terminal fragment (CTF25), in addition to other minor CTFs and the full-length TDP-43, in cytoplasmic aggregates. CTFs are hallmarks of forms of disease whose pathology is associated exclusively with abnormalities in TDP-43 and are observed in patients with not only ALS and FTLD but also Alzheimer's disease 5 . Amino-terminal (N-terminal) analysis of CTFs obtained from brain autopsies of patients with FTLD has resulted in the identification of three cleavage sites, Arg208, Asp219 and Asp247, which give rise to CTFs 6,7 . However, the expected sizes of the resulting CTFs are less than 25 kDa, which suggests that CTF25 is probably cleaved at a different position.TDP-43 is a widely expressed 414-amino acid (a.a.) protein comprised of two RNA recognition motifs (RRM1 at a.a. 106-176 and RRM2 at a.a. 191-262) and the GRD 8,9 . The chronic stabilization...

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TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes

Cited by 361 publications

References 32 publications

RNA–protein analysis using a conditional CRISPR nuclease

RNA–protein analysis using a conditional CRISPR nuclease

Folding of the RNA Recognition Motif (RRM) Domains of the Amyotrophic Lateral Sclerosis (ALS)-linked Protein TDP-43 Reveals an Intermediate State

The cleavage pattern of TDP-43 determines its rate of clearance and cytotoxicity

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