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).
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 9. Abstract
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