Hu Antigen R (HuR) Is a Positive Regulator of the RNA-binding Proteins TDP-43 and FUS/TLS: Implications for Amyotrophic Lateral Sclerosis

Liang Lu,Lei Zheng,Ying Si,Wenyi Luo,Gwendal Dujardin,Thaddaeus Kwan,Nicholas R Potochick,Sunnie R Thompson,David A Schneider,Peter H King

doi:10.1074/jbc.m114.573246

Liang Lu, Lei Zheng + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m114.573246

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Abstract

Posttranscriptional gene regulation is governed by a network of RNA-binding proteins (RBPs) that interact with regulatory elements in the mRNA to modulate multiple molecular processes, including splicing, RNA transport, RNA stability, and translation. Mounting evidence indicates that there is a hierarchy within this network whereby certain RBPs cross-regulate other RBPs to coordinate gene expression. HuR, an RNA-binding protein we linked previously to aberrant VEGF mRNA metabolism in models of SOD1-associated amyotrophic lateral sclerosis, has been identified as being high up in this hierarchy, serving as a regulator of RNA regulators. Here we investigated the role of HuR in regulating two RBPs, TDP-43 and FUS/TLS, that have been linked genetically to amyotrophic lateral sclerosis. We found that HuR promotes the expression of both RBPs in primary astrocytes and U251 cells under normal and stressed (hypoxic) conditions. For TDP-43, we found that HuR binds to the 3' untranslated region (UTR) and regulates its expression through translational efficiency rather than RNA stability. With HuR knockdown, there was a shift of TDP-43 and FUS mRNAs away from polysomes, consistent with translational silencing. The TDP-43 splicing function was attenuated upon HuR knockdown and could be rescued by ectopic TDP-43 lacking the 3' UTR regulatory elements. Finally, conditioned medium from astrocytes in which HuR or TDP-43 was knocked down produced significant motor neuron and cortical neuron toxicity in vitro. These findings indicate that HuR regulates TDP-43 and FUS/TLS expression and that loss of HuR-mediated RNA processing in astrocytes can alter the molecular and cellular landscape to produce a toxic phenotype.

Highlights

RNA processing abnormalities have been linked to amyotrophic lateral sclerosis (ALS)
For TDP-43, we found that Hu antigen R (HuR) binds to the 3؅ untranslated region (UTR) and regulates its expression through translational efficiency rather than RNA stability
HuR Regulates TDP-43 and FUS/TLS—We used shRNA to knock down HuR in U251 clones expressing mutant or wildtype superoxide dismutase 1 (SOD1) and in primary astrocytes [14, 28]

Summary

Background

RNA processing abnormalities have been linked to amyotrophic lateral sclerosis (ALS). The genetic linkage of TAR DNA binding protein 43 (TDP43) and fused in sarcoma/translocated in liposarcoma (FUS/ TLS) with amyotrophic lateral sclerosis (ALS) has elevated RNA processing to the forefront as a potential mechanism of motor neuron degeneration in ALS [1] These RNA binding proteins contain one or more RNA recognition motifs (RRM) that bind to target mRNA ligands to direct splicing, RNA transport, stabilization, and other processes. Suppression of HuR in primary astrocytes leads to the production of soluble factors that are toxic to cortical and motor neurons, similar to when TDP-43 is directly suppressed These findings provide evidence that HuR plays a role in regulating ALS-associated RNA binding proteins and raise the possibility that HuR may contribute to motor neuron disease pathology by its unchecked positive regulation of ALSassociated TDP-43 and FUS/TLS

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION