Neuronal Elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability - PubMed (original) (raw)

. 2012 Sep 20;75(6):1067-80.

doi: 10.1016/j.neuron.2012.07.009.

Hirotaka J Okano, Kirk B Jensen, Woong-Yang Park, Ru Zhong, Jernej Ule, Aldo Mele, John J Fak, Chingwen Yang, Chaolin Zhang, Jong Yoo, Margaret Herre, Hideyuki Okano, Jeffrey L Noebels, Robert B Darnell

Affiliations

Neuronal Elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability

Gulayse Ince-Dunn et al. Neuron. 2012.

Abstract

The paraneoplastic neurologic disorders target several families of neuron-specific RNA binding proteins (RNABPs), revealing that there are unique aspects of gene expression regulation in the mammalian brain. Here, we used HITS-CLIP to determine robust binding sites targeted by the neuronal Elav-like (nElavl) RNABPs. Surprisingly, nElav protein binds preferentially to GU-rich sequences in vivo and in vitro, with secondary binding to AU-rich sequences. nElavl null mice were used to validate the consequence of these binding events in the brain, demonstrating that they bind intronic sequences in a position dependent manner to regulate alternative splicing and to 3'UTR sequences to regulate mRNA levels. These controls converge on the glutamate synthesis pathway in neurons; nElavl proteins are required to maintain neurotransmitter glutamate levels, and the lack of nElavl leads to spontaneous epileptic seizure activity. The genome-wide analysis of nElavl targets reveals that one function of neuron-specific RNABPs is to control excitation-inhibition balance in the brain.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Fig. 1

Fig. 1. Generation of _Elavl_3-/- knockout mice

(A) The targeting construct used in generating elavl3 KO locus by homologous recombination. (B) The expression of Elavl3 protein is abolished in _Elavl3_-/- brain tissue (P21 WT, heterozygote and KO mice (littermates), as indicated). The lower heavy band corresponds to Elavl3, upper bands represent Elavl2 and Elavl4. Results from P21 WT, Elavl3+/- and _Elavl3_-/- mice were repeated in 3 independent litters. (C) Rotarod or hotplate testing of cerebellar or sensory physiology in Elavl3+/- or -/- littermates, as indicated; second until falling off the rod or tail twitch are shown. Rotarod testing was done with 6~8 week-old males (n=3; p < 0.0001), and hotplate testing was done with 7~9 week-old males (n=3, p = 0.11). (D-E) IF microscopy of _Elavl3_-/- mice compared to WT (+/+) littermate controls. A pan anti-nElavl antibody (α-nElavl) was used for IF, and contrasted with staining for the Purkinje neuronal marker Calbindin (D) or nuclei (DAPI, (E)). (E) Arrows point to nElavl immunostaining in WT and lack of it in Elavl3-/- DG.

Fig. 2

Fig. 2. Isolation of nElavl-RNA complexes by CLIP

(A) nElavl-RNA complexes from WT and Elavl3-/- forebrain tissue from mice at age P0 were UV-crosslinked and immunoprecipitated (by nElavl antibody1) using the CLIP method. Representative autoradiograms of [γ-32P]ATP 5’end labeled RNA molecules, run on a polyacrylamide gel and blotted onto nitrocellulose filter are shown. Overdigestion of the lysate with RNase A (1:100 dilution) resulted in approximately a 40 kDa band, corresponding to nElavl and associated RNA fragments that are protected. The size of nElavl-associated RNA was titrated by increasing dilutions of RNase A treatment. Stronger signal was detected in the WT lanes as opposed to Elavl3-/- lanes. The signal detected in the Elavl3-/- lanes are due to Elavl2/4 that are also immunoprecipitated by the same antibody. Hatched box marks the piece of membrane from which nElavl-associated RNA was isolated. (B, C and D) Line traces of nElavl-RNA membrane shown in (A) are plotted. Individual lanes are color-coded. (E) No signal was detected when two different control antibodies (anti-Yo autoantibodies) were used (lanes 1-4) or when UV-crosslinking was omitted (lanes 7-8). nElavl-RNA complexes have been immunoprecipitated using antibody2. See also Fig.S1 and Tables S1-3.

Fig. 3

Fig. 3. nElavl binds to U-rich sequences

(A) Distribution of nElavl tag clusters generated from 6 independent WT tissue samples is plotted as a function of biologic complexity. (B-C) Cluster sequences with either FDR<0.01 (B) or BC>=1. (C) were used to predict nElavl binding motif by MEME-CHIP tool. (D) Top ten most frequent hexamers found in nElavl clusters (FDR<0.01). See also Tables S4-6.

Figure 4

Figure 4. In vitro selection of nElavl binding RNA molecules

(A) Representative results from in vitro RNA selection with the nElavl proteins. RNA selection was carried out using the Elavl2, Elavl3 or Elavl4 proteins for 6-8 rounds of selection (R6-R8, as indicated). Consensus GU rich elements are shown in blue and below the diagram. (B) RNA gel-shift assay, in which DR8-9 clone RNA selected by Elavl4 was incubated with recombinant Elavl4 as indicated. Multiple forms of Elavl4/RNA complexes (arrows) have slower migration profiles with increasing amounts of protein (RNA=50 fmol/lane, Protein=0, 25, 50 or 75 ng), and this effect is specific, as no effect on RNA mobility was seen with either hnRNP A1 (25 ng), or when Elavl4 was incubated with an irrelevant control RNA (SB2, a 52 base NOVA1 consensus sequence). (C) Results of filter binding assays in which the indicated amounts of Elavl4 fusion protein were incubated with radiolabeled selected RNA (where the number of GUUGU repeats (n) is shown, see (A)). Red: DR8-2 RNA; Blue: DR8-15 RNA; Green: DR7-5 RNA; Black: control SB2 RNA (50 fmol/reaction). Estimated kD’s are shown.

Fig. 5

Fig. 5. Normalized complexity map for nElavl-dependent alternative splicing

(A) nElavl tags mapping to nElavl-regulated cassette exons or flanking introns are plotted onto a composite transcript as a function of distance to the 5’ or 3’ junctions of the alternative exon. Tags from independent CLIP experiments are color-coded. Red and grey boxes represent a generic alternative cassette exon and flanking constitutive exons, respectively. (B) Normalized complexity map of nElavl-dependent alternative splicing of cassette exons. Red and blue peaks represent binding associated with nElavl-dependent exon inclusion and exclusion, respectively. Motif preferences of 250 nt sequences flanking nElavl-regulated alternative exons are shown. (C) Motif preferences of 250 nt sequences flanking randomly selected alternative exons that display no change in isoform abundance in DKO mice are shown. See also Fig.S2 and Table S8.

Fig. 6

Fig. 6. nElavl-dependent regulation of the brain enzyme glutaminase

(A) The two mRNA isoforms of the glutaminase gene (Gls) and nElavl binding sites are shown. Individual colors depict different experiments. Alternative use of a 3’ splice site generates two Gls isoforms with different 3’ terminal coding sequences and 3’ UTRs. Gls-s and Gls-l refer to short and long isoforms, respectively. (B) RT-PCR amplification of the two Gls isoforms in WT and Elavl3-/-;Elavl4-/- cortex of age P0 mice. (C) Western blot analysis of the two Gls isoforms in littermate WT and Elavl3-/-;Elavl4-/- cortex of age P0 mice. Each lane represents an independent mouse. (D) Q-PCR quantification of the abundance of two Gls mRNA isoforms in littermate WT and Elavl3-/-;Elavl4-/- cortex of age P0 mice. (E) Quantification of data shown in panel (C). (F) Quantification of total glutamate levels in cortex of 3 WT and 3 Elavl3-/-;Elavl4-/- littermate age P0 mice are presented. Glutamate levels in WT samples are normalized to 100% in the Y-axis. * denotes p<0.01 (t-test). Error bars denote standard deviation. See also Fig.S3-4 and Table S9.

Fig. 7

Fig. 7. EEG Analysis of cortical function in _Elavl3_-/- mice

Spontaneous bilateral EEG activity recorded from awake and behaving 3-6 month old adult (A) WT, (B) Elavl3+/-, and (C) Elavl3-/- mice. Cortical recordings are displayed from left (L-reference) and right (R-reference) hemisphere temporoparietal electrodes. WT mice lack abnormal discharges seen occurring intermittently in Elavl3+/- and _Elavl3_-/- mice. Brief seizures shown in Elavl3-/- mice are accompanied by mild convulsive clonic movements. Seizures were detected in both Elavl3-/- and -/+ mice. Calibration, 1 sec (A and C, 0.5 sec in B), 200 microvolts (A-C). See also Fig.S5-6, Tables S10-11 and Movie S1.

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