Slicing and Dicing: TDP-43 Teams Up With Nucleases to Make MicroRNAs

TDP-43 shepherds select microRNAs through their maturation process from nuclear transcripts to grown-up regulatory fragments set loose in the cytoplasm, according to a paper in the February 9 Proceedings of the National Academy of Sciences online. “Our work is the first evidence that TDP-43 directly regulates miRNA processing,” wrote first and senior author Yukio Kawahara, of Osaka University in Japan in an e-mail to ARF. TDP-43, he found, interacts with specific miRNAs at two different stages of their development, to promote neuronal outgrowth and, perhaps, mediate other cellular processes. Mutations in TDP-43 that cause amyotrophic lateral sclerosis, Kawahara predicted, could interrupt this activity and slash expression of its target miRNAs.

The making of an miRNA is a multistage process. Newborn transcripts, called pri-miRNAs, are cleaved in the nucleus by the Drosha RNase complex to form pre-miRNAs. These graduate to the cytoplasm, where another RNase conglomerate made up of Dicer and associates cleave them again, forming the mature miRNA. The microRNA then interacts with other proteins to bind its target mRNA, causing degradation or inhibiting translation.

Although TDP-43’s precise roles in health and disease are uncertain, it appears to regulate RNAs (reviewed in Warraich et al., 2010; see ARF related news story). The protein pals around with Drosha (Gregory et al., 2004; see ARF related news story on Ling et al., 2010), which inspired Kawahara to investigate its role in miRNA processing. Researchers previously linked TDP-43 to levels of two miRNAs, let-7b and miR-663, in hepatoma cells (Buratti et al., 2010). MicroRNAs have been linked to a motor neuron degeneration phenotype in Dicer knockout mice (see ARF related news story on Haramati et al., 2010), and lack of miR-206 in muscles causes ALS model mice to succumb more quickly than normal (see ARF related news story on Williams et al., 2009).

In co-immunoprecipitation experiments in HEK293T human embryonic kidney cell cultures, Kawahara confirmed the interaction between Drosha and TDP-43. He also observed that TDP-43 interacted with Dicer in the cytoplasm. Since Kawahara presumed that TDP-43 must promote miRNA processing, he used microarrays to look for miRNAs affected by the protein. He applied RNA interference to get rid of TDP-43 in cultures of SH-SY5Y human neuroblastoma cells, HeLa human cervical cancer cells, and Neuro2a mouse neuroblastoma cells. With the microarrays, he identified miRNAs that dropped by more than 1.5-fold in the absence of TDP-43 (no miRNA expression increased in the absence of TDP-43). Using quantitative reverse transcriptase-polymerase chain reaction, Kawahara verified that loss of TDP-43 diminished miR-132, miR-143, miR-574, and miR-558. He confirmed, using immunoprecipitation and gel shift assay cells, that TDP-43 binds these pri-miRNAs, but not other pri-miRNAs, in SH-SY5Y cells. In the cytoplasm, TDP-43 bound only two of the pre-miRNAs, miR-143 and miR-574, suggesting it is only needed to help Dicer process these two RNAs.

MicroRNA-143 is involved in various jobs including smooth muscle development (Cordes et al., 2009) and tumor suppression (Kent et al., 2010), while miR-558 is promotes tumor growth (Shohet et al., 2011). MicroRNA-574 has been linked to stress responses, and it regulates expression of β-glycan, a growth factor receptor (Morgan and Bale, 2011). MicroRNA-132 is highly expressed in the central nervous system, Kawahara noted, where it regulates dendritic outgrowth (Vo et al., 2005; Magill et al., 2010).

Kawahara examined the interaction between TDP-43 and miR-132 in Neuro2a cells that he induced to differentiate. TDP-43 knockdown minimized neurite outgrowth, but artificially providing excess miR-132 restored it. This suggests that the stumpy outgrowth was due, in part, to a lack of TDP-43 support for miR-132 maturation. “TDP-43, as a component of not only nuclear Drosha complex but also cytoplasmic Dicer complex, promotes neuronal outgrowth by facilitating biogenesis of specific miRNAs,” Kawahara concluded. “Although a number of roles have been identified for TDP-43 in RNA metabolism and processing in the nucleus, I would like to suggest that TDP-43 also plays a pivotal role in RNA processing in the cytoplasm,” he added.

Using TDP-43 deletion mutants, Kawahara determined that the carboxyl-terminal portion of TDP-43 is crucial to interact with the miRNA-processing complexes. “TDP-43 mutations are highly concentrated in this region,” he wrote. “Therefore, it is possible that those mutations reduce the expression of TDP-43-regulated miRNAs by weakening the interaction [of TDP-43] with Drosha or Dicer complexes.” To further examine the role of TDP-43 and miRNAs in neurodegenerative diseases—such as ALS and frontotemporal dementia—Kawahara plans to study the effects of TDP-43 mutations on miRNA maturation and identify miRNAs expressed in motor and frontotemporal neurons.

The work could have implications for not only ALS caused by TDP-43 mutations, but also sporadic ALS, since TDP-43 builds up in cytoplasmic aggregates in both instances. “Accumulations of TDP-43 in inclusions during neurodegenerative disorders may prevent its functional role in miRNA biogenesis,” noted Pierre Lau Poui Cheung of KU Leuven, Belgium in an e-mail to ARF (see full comment below).—Amber Dance

Comments

  1. This work of Yukio Kawahara and Ai Mieda-Sato is very interesting. It complements previous work from the groups of Don Cleveland, Bob Brown, and many others linking RNA metabolism to the pathology of TDP-43 in FTD and ALS. It also provides a strong clue that miRNAs may be involved in TDP-43 associated neurodegeneration, as also suggested by Francisco Baralle and myself (see Buratti et al., 2010 and Haramati et al., 2010).

    The study here reveals facets of the molecular mechanism by which TDP-43 functions in regulation of miRNA biogenesis, both in the nucleus (with Drosha) and in the cytoplasm (with Dicer).

    I like to think of several questions emerging from this work:

    1. In what way do ALS-causing TDP-43 mutations impair TDP-43's interaction with Dicer or Drosha in vivo?

    2. What part of TDP-43-associated pathology involves regulation of miRNA bioprocessing versus other events (including, e.g., splicing)?

    3. What miRNAs are actually regulated by TDP-43 in relevant populations of authentic neurons (e.g., in the frontal cortex and motor neurons)?

    4. What pathways do miRNA genes downstream of TDP-43 control?

    All these make major challenges for follow-up work.

    References:

    Buratti E, De Conti L, Stuani C, Romano M, Baralle M, Baralle F. Nuclear factor TDP-43 can affect selected microRNA levels. FEBS J. 2010 May;277(10):2268-81. PubMed.

    Haramati S, Chapnik E, Sztainberg Y, Eilam R, Zwang R, Gershoni N, McGlinn E, Heiser PW, Wills AM, Wirguin I, Rubin LL, Misawa H, Tabin CJ, Brown R, Chen A, Hornstein E. miRNA malfunction causes spinal motor neuron disease. Proc Natl Acad Sci U S A. 2010 Jul 20;107(29):13111-6. PubMed.

    View all comments by Eran Hornstein

  2. The regulation of miRNA biogenesis by TAR DNA-binding protein 43 (TDP-43) represents a bridge between neurodegeneration and regulation of genes at the post-transcriptional level. TDP-43 is the major constituent of inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-TDP). Under physiological conditions, TDP-43 is localized in the nucleus of cells. This paper shows that TDP-43 can physically associate with the Drosha complex in the nucleus to alter expression of miRNAs in two cell lines, while knockdown of TDP-43 leads to reduction of at least six miRNAs. Under pathological conditions, TDP-43 is relocated to the cytoplasm and accumulates in inclusions. Since the paper also shows that TDP-43 can interact with the Dicer complex to promote production of a subset of miRNAs, accumulation of TDP-43 in inclusions during neurodegenerative disorders may prevent its functional role in miRNA biogenesis, leading to alteration of miRNA expression. For instance, TDP-43 depletion in Neuro2A cells led to a reduced level of miR-132 and attenuation of neurite outgrowth. This raises the possibility that TDP-43 inclusions as a secondary histopathological feature in other neurodegenerative disorders, such as Alzheimer’s disease, may also lead to alteration of miRNAs.

    Based on our experience, a subset of miRNAs in late-onset Alzheimer’s disease is affected from early to late stages of disease. Is there an overlap of altered miRNAs in Alzheimer’s disease, ALS, and FTLD that can mechanistically define pathways and networks involved in aging and neuronal homeostasis?

    There is now strong belief that we have the appropriate toolbox to study miRNAs and identify their targets, thus advancing our understanding of molecular mechanisms of neurodegeneration.

    View all comments by Pierre Lau Poui Cheung

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References

News Citations

  1. Toxic TDP-43 Too Tough to Degrade, Plays Prion?
  2. Motor Neuron Disease Risk Factors: Fresh miRNA Clue, KIFAP3 Letdown
  3. Muscle MicroRNA Repairs Nerve-Muscle Connection in ALS Model

Paper Citations

  1. Warraich ST, Yang S, Nicholson GA, Blair IP. TDP-43: a DNA and RNA binding protein with roles in neurodegenerative diseases. Int J Biochem Cell Biol. 2010 Oct;42(10):1606-9. PubMed.
  2. Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R. The Microprocessor complex mediates the genesis of microRNAs. Nature. 2004 Nov 11;432(7014):235-40. PubMed.
  3. Ling SC, Albuquerque CP, Han JS, Lagier-Tourenne C, Tokunaga S, Zhou H, Cleveland DW. ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc Natl Acad Sci U S A. 2010 Jul 27;107(30):13318-23. PubMed.
  4. Buratti E, De Conti L, Stuani C, Romano M, Baralle M, Baralle F. Nuclear factor TDP-43 can affect selected microRNA levels. FEBS J. 2010 May;277(10):2268-81. PubMed.
  5. Haramati S, Chapnik E, Sztainberg Y, Eilam R, Zwang R, Gershoni N, McGlinn E, Heiser PW, Wills AM, Wirguin I, Rubin LL, Misawa H, Tabin CJ, Brown R, Chen A, Hornstein E. miRNA malfunction causes spinal motor neuron disease. Proc Natl Acad Sci U S A. 2010 Jul 20;107(29):13111-6. PubMed.
  6. Williams AH, Valdez G, Moresi V, Qi X, McAnally J, Elliott JL, Bassel-Duby R, Sanes JR, Olson EN. MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice. Science. 2009 Dec 11;326(5959):1549-54. PubMed.
  7. Cordes KR, Sheehy NT, White MP, Berry EC, Morton SU, Muth AN, Lee TH, Miano JM, Ivey KN, Srivastava D. miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature. 2009 Aug 6;460(7256):705-10. PubMed.
  8. Kent OA, Chivukula RR, Mullendore M, Wentzel EA, Feldmann G, Lee KH, Liu S, Leach SD, Maitra A, Mendell JT. Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway. Genes Dev. 2010 Dec 15;24(24):2754-9. PubMed.
  9. Shohet JM, Ghosh R, Coarfa C, Ludwig A, Benham AL, Chen Z, Patterson DM, Barbieri E, Mestdagh P, Sikorski DN, Milosavljevic A, Kim ES, Gunaratne PH. A genome-wide search for promoters that respond to increased MYCN reveals both new oncogenic and tumor suppressor microRNAs associated with aggressive neuroblastoma. Cancer Res. 2011 Jun 1;71(11):3841-51. PubMed.
  10. Morgan CP, Bale TL. Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage. J Neurosci. 2011 Aug 17;31(33):11748-55. PubMed.
  11. Vo N, Klein ME, Varlamova O, Keller DM, Yamamoto T, Goodman RH, Impey S. A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc Natl Acad Sci U S A. 2005 Nov 8;102(45):16426-31. PubMed.
  12. Magill ST, Cambronne XA, Luikart BW, Lioy DT, Leighton BH, Westbrook GL, Mandel G, Goodman RH. microRNA-132 regulates dendritic growth and arborization of newborn neurons in the adult hippocampus. Proc Natl Acad Sci U S A. 2010 Nov 23;107(47):20382-7. PubMed.

Other Citations

  1. ARF related news story

Further Reading

Primary Papers

  1. Kawahara Y, Mieda-Sato A. TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes. Proc Natl Acad Sci U S A. 2012 Feb 28;109(9):3347-52. PubMed.

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