Gemin5 Binds to the Survival Motor Neuron mRNA to Regulate SMN Expression

Abstract

Reduced expression of SMN causes spinal muscular atrophy, a severe neurodegenerative disease. Despite the importance of maintaining SMN levels, relatively little is known about the mechanisms by which SMN levels are regulated. We show here that Gemin5, the snRNA-binding protein of the SMN complex, binds directly to the SMN mRNA and regulates SMN expression. Gemin5 binds with high specificity, both in vitro and in vivo, to sequence and structural elements in the SMN mRNA 3'-untranslated region that are reminiscent of the snRNP code to which Gemin5 binds on snRNAs. Reduction of Gemin5 redistributes the SMN mRNA from heavy polysomes to lighter polysomes and monosomes, suggesting that Gemin5 functions as an activator of SMN translation. SMN protein is not stoichiometrically present on the SMN mRNA with Gemin5, but the mRNA-binding activity of Gemin5 is dependent on SMN levels, providing a feedback mechanism for SMN to regulate its own expression via Gemin5. This work both reveals a new autoregulatory pathway governing SMN expression, and identifies a new mechanism through which SMN can modulate specific mRNA expression via Gemin5.

Keywords: Gemin5; RNA-binding protein; SMA; SMN; mRNA; neurodegenerative disease; post-transcriptional regulation; spinal muscular atrophy; survival motor neuron; translation.

FIGURE 1.

Gemin5 binds to the mature SMN mRNA in vivo and in vitro. RT-PCR of modified PAR-CLIP to detect SMN mRNA bound to Gemin5 in both (A) HeLa and (B) MN1 cell extract. Control is an isotype-matched IgG antibody. C, diagram of RNA transcripts used in D and E. The SMN pre-mRNA construct is 192 nucleotides in length and 118 nucleotides for the mature mRNA exclusive of the 5 nucleotide pA tail. The regions deleted in subsequent experiments are indicated by H2, H1, as well as the U-rich region (white text). Further 10 or 20 nucleotide deletions were made to the 3′-end as indicated. D, Western blot of mutant and wild type SMN mRNA fragments in streptavidin pulldown assays. Control RNA used is a 60-nucleotide nonspecific RNA transcribed from empty pSP72. E, (left) Western blot of mutant and wild type SMN probing for proteins of the SMN complex in a streptavidin pulldown assay. Right, Western blot of SMN immunoprecipitation showing expected ratios of complex proteins. Control is the same as in D. For all, input represents extract from cell lines. IP, immunoprecipitation; Δ10 and Δ20, deletion of 10 or 20 nucleotides; wt, wild type; H2, helical region 2; H1, helical region 1.

FIGURE 2.

Gemin5 directly binds to the SMN mRNA. A, direct RNA binding assay performed with radiolabeled wild type SMN 3′-UTR or a control RNA sequence (as described in Fig. 1D). The RNA was bound to immunopurified Gemin5, Gemin3 or SMN protein or to a nonspecific isotype matched control IgG antibody. B, direct RNA binding assay as in A performed with 10 or 20 nucleotide truncations of the SMN 3′-UTR. For both, input represents 1% or 1,000 CPM of RNA used in each binding assay.

FIGURE 3.

Gemin5 binds to specific sequence and structure elements of the SMN 3′-UTR. A, mfold structure prediction for the 3′ fragment of the SMN 3′-UTR. B, RNA binding assay performed with radiolabeled SMN RNA that contain point mutations in the AU sequence as indicated above each lane. C, RNA binding assay performed with stem-loop deleted SMN RNA as indicated in A. Input and control are the same as described in Fig. 2. H1, helical region 1; H2, helical region 2; wt, wild type.

FIGURE 4.

Gemin5 regulates expression of endogenous and reporter SMN. A, Western blot of protein from HeLa cells stably expressing shRNA targeting GFP (control) or Gemin5. B, quantitation of protein in A using ImageStudio for Li-Cor and normalizing signals to the loading control tubulin. The signal is expressed as a percentage of the signal in the control RNAi cells. C, dual luciferase reporter assay with a control or SMN 3′-UTR in the presence of transiently transfected control (nonspecific nontargeting siRNA) or Gemin5 siRNA. The control on the x-axis represents a luciferase expressed without an inserted 3′-UTR sequence. Ctrl, control; *, p < 0.05.

FIGURE 5.

Knockdown of Gemin5 does not affect SMN RNA stability or turnover, but does affect translation. A, real-time quantitative PCR of SMN in RNA extracts from control or Gemin5 RNAi cells. B, real-time quantitation of SMN RNA remaining after treatment with actinomycin D in cells with or without Gemin5 knockdown. C, UV absorbance trace of polysome fractions obtained from a sucrose gradient of cell lysates from control or Gemin5 knockdown cells. D, real-time PCR quantitation of SMN RNA in odd fractions of polysome gradient shown in C. For all, control RNAi represents a nonspecific nontargeting siRNA. Ctrl, control; *, p < 0.05.

FIGURE 6.

Knockdown of SMN causes Gemin5-dependent up-regulation of SMN reporter. A, Western blot of streptavidin pulldown assay using cell extracts from control (nontargeting siRNA) or SMN knockdown cells. Control RNA used is nonspecific RNA transcribed from an empty pSP72 vector. Input is 10 μg of total protein from respective cell lines. B, dual luciferase reporter assay performed with a wild type SMN RNA (SMN 3′-UTR) or a Gemin5 binding sequence-deleted mutant of SMN (SMN mut 3′-UTR) expressed in cells also transfected with control or SMN RNAi. Ctrl, control; mut, mutant, *, p < 0.05.