Alternative mRNA splicing of SMRT creates functional diversity by generating corepressor isoforms with different affinities for different nuclear receptors

Abstract

Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts.

FIG. 1. Schematic representation of SMRT isoforms

A, the amino acid sequence of the SMRTα exon and surrounding sequences. The CoRNR box sequence of the SMRT S1 domain is indicated in boldface and boxed. The numbers indicate the amino acid positions in SMRTα. B, schematic alignment of the domains of SMRTα and SMRTτ as well as N-CoR. Black boxes indicate the repression domains, and vertical bars indicate the positions of the CoRNR box sequences within each receptor interaction domain. The amino acid positions for each isoform are indicated. C, EST clones that contain sequences identical to either SMRTα or SMRTτ and the library tissue sources for each EST. D, schematic representation of the alternative mRNA splicing events that give rise to SMRTα and SMRTτ. The nucleotide positions within the open reading frames of SMRTα and SMRTτ are indicated. Nucleotide numbers within the human genome chromosome 12 HS12_9912 segment sequence are also indicated (GenBank^™/EBI accession number NT_009755).

FIG. 2. Protein accumulation and nuclear localization of SMRTα and SMRTτ

A, Western blot of CV-1 cells either untransfected or transfected with plasmids expressing Myc-tagged full-length SMRTα or Myc-tagged SMRTτ. B, fluorescent micrograph of CV-1 cells transfected with plasmids expressing GFP, GFP-SMRTα, or GFP-SMRTτ. C, change in the subcellular localization of SMRTα or SMRTτ in response to MEKK1.

FIG. 3. EMSA interaction between TRβ1 and the SMRT S1 domains

A, human TRβ1 derived from a recombinant baculovirus/Sf9 cell system (T) or equivalent non-recombinant preparations (N) were incubated with radiolabeled DR4 oligonucleotide DNA and GST (G), GST-SMRTα(S1) (α), or GST-SMRTτ(S1) (τ). Anti-TRβ1 antibody (Ab; catalog no. MA1–215, Affinity BioReagents, Golden, CO), 1 µM T₃, and/or human RXRα (from baculovirus/Sf9 preparations) was also added to certain samples as indicated. B, varying amounts of purified GST-SMRTα(S1) or GST-SMRTτ(S1) protein were added to a constant amount of TRβ1 protein and radiolabeled DR4 probe. C, the TRβ1·DNA complexes supershifted to a slower mobility by the SMRTα or SMRTτ S1 domain (i.e. bound by SMRTα or SMRTτ) were quantified relative to the amount of SMRT protein added to each binding reaction. From these data, the apparent dissociation constants for both the SMRTα and SMRTτ S1 domains were determined. The graph represents the mean of n > 3 replicates. Error bars indicate S.E. RID, receptor interaction domain.

FIG. 4. Interaction between SMRT receptor interaction domains and TRα1 or TRβ1 on a DR4 DNA element.

Varying amounts of purified GST-SMRTα(S1) or GST-SMRTτ (S1) were added to binding reactions containing TRβ1 (A), or TRα1 (C) together with a radiolabeled DR4 oligonucleotide probe. Alternatively, varying amounts of purified GST-SMRTα(S1/S2) or GST-SMRTτ(S1/S2) were added to binding reactions containing TRβ1 (B) or TRβ1 (D) together with the radiolabeled DR4 oligonucleotide probe. The receptor-DNA complexes supershifted by addition of the SMRT constructs were quantified relative to the amount of SMRT protein added to each binding reaction. Error bars indicate S.E. of three replicate experiments. RID, receptor interaction domain.

FIG. 5. Interaction between SMRT receptor interaction domains and TRα1 or TRβ1 on a DIV6 DNA element

Varying amounts of purified GST-SMRTα(S1) or GST-SMRTτ(S1) were added to binding reactions containing TRβ1 (A) or TRα1 (C) together with a radiolabeled DIV6 oligonucleotide probe. Alternatively, varying amounts of purified GST-SMRTα(S1/S2) or GST-SMRTτ(S1/S2) were added to binding reactions containing TRβ1 (B) or TRα1 (D) together with the radiolabeled DIV6 oligonucleotide probe. The receptor-DNA complexes supershifted by addition of the SMRT constructs were quantified relative to the amount of SMRT protein added to each binding reaction. Error bars indicate S.E. of two or more replicate experiments. RID, receptor interaction domain.

FIG. 6. Interaction between SMRT receptor interaction domains and RXRα/TR heterodimers or RARα homodimers

Varying amounts of purified GST-SMRTα(S1/S2) or GST-SMRTτ(S1/S2) were added to binding reactions containing TRα1 and RXRα (A) or TRβ1 and RXRα (B) and a DR4 oligonucleotide probe. Alternatively, varying amounts of purified GST-SMRTα(S1) or GSTSMRT α(S1) (C), GST-SMRT(S2) (D), or GST-SMRTα(S1/S2) or GST-SMRTτ(S1/S2) (E) were added to binding reactions containing RARα and a radiolabeled DR5 oligonucleotide probe. The receptor·DNA complexes supershifted by addition of the SMRT constructs were quantified relative to the amount of SMRT protein added to each binding reaction. Error bars indicate S.E. of three replicate experiments. RID, receptor interaction domain.

FIG. 7. Disruption of SMRT·TR complexes with thyroid hormone

A fixed amount of purified GST-SMRTα(S1) or GST-SMRTτ(S1) were added to binding reactions containing TRβ1 (A) or TRα1 (C) together with a radiolabeled DR4 oligonucleotide probe. Alternatively, a fixed amount of purified GST-SMRTα(S1/S2) or GST-SMRTτ(S1/S2) were added to binding reactions containing TRβ1 (B) or TRα1 (D) together with the radiolabeled DR4 probe. Varying amounts of T₃ were added to each reaction. The SMRT-TR-DNA complexes formed in the presence of the differing T₃ concentrations were quantified. (SMRT-TR-DNA complexes observed in the absence of hormone were defined as 100%.) Error bars for each data point indicate S.E. (n = 3).

FIG. 8. Dominant-negative inhibition of TRβ1-mediated repression

CV-1 cells were transfected with a TRβ1 expression vector, the lysozyme F2 element-luciferase reporter, and varying amounts of either a Myc-SMRTα(S1/S2) or Myc-SMRTτ(S1/S2) expression vector. Transfected cells were analyzed for luciferase activity. Luciferase activity for each sample is plotted versus the amount of SMRT expression vector. The unrepressed level of luciferase activity (no TRβ1) is indicated. Error bars indicate S.E. of three replicate experiments. RID, receptor interaction domain.

FIG. 9. Analysis of expression of SMRTα and SMRTτ in various mouse tissues

A, cDNAs from heart (H), brain (B), kidney (K), spleen (S), liver (Li), lung (Lu), testis (T), or skeletal muscle (M) were amplified with primers that span the SMRTα exon; these are expected to produce a 442-bp product for SMRTα and a 301-bp product for SMRTτ. SMRT samples were amplified for 30 cycles. The cDNA from the same mouse tissues was also amplified for 20 cycles using a primer for GAPDH, which produces a 125-bp product. B, samples from duplicate reactions from two mice were analyzed using an Alpha Innotech FluorChem 8900 densitometer. Averages of the ratio of SMRTα to SMRTτ and S.E. for each are plotted (n > 3).