CELF6, a Member of the CELF Family of RNA-binding Proteins, Regulates Muscle-specific Splicing Enhancer-dependent Alternative Splicing

Abstract

We previously described a family of five RNA-binding proteins: CUG-binding protein, embryonic lethal abnormal vision-type RNA-binding protein 3, and the CUGbinding protein and embryonic lethal abnormal visiontype RNA-binding protein 3-like factors (CELFs) 3, 4, and 5. We demonstrated that all five of these proteins specifically activate exon inclusion of cardiac troponin T minigenes in vivo via muscle-specific splicing enhancer (MSE) sequences. We also predicted that a sixth family member, CELF6, was located on chromosome 15. Here, we describe the isolation and characterization of CELF6. Like the previously described CELF proteins, CELF6 shares a domain structure containing three RNA-binding domains and a divergent domain of unknown function. CELF6 is strongly expressed in kidney, brain, and testis and is expressed at very low levels in most other tissues. In the brain, expression is widespread and maintained from the fetus to the adult. CELF6 activates exon inclusion of a cardiac troponin T minigene in transient transfection assays in an MSE-dependent manner and can activate inclusion via multiple copies of a single element, MSE2. These results place CELF6 in a functional subfamily of CELF proteins that includes CELFs 3, 4, and 5. CELF6 also promotes skipping of exon 11 of insulin receptor, a known target of CELF activity that is expressed in kidney.Alternative splicing allows the production of multiple mRNA species from a single gene, which often give rise to functionally distinct protein isoforms. Regulation of alternative splicing not only produces multiple mRNAs but can also modulate the levels of these different isoforms in a tissue-or developmental stage-specific manner to meet the functional needs of the cell. Numerous examples of regulated alternative splicing have been found, but few regulatory factors that control cell-specific alternative splicing have been identified. In the best-characterized vertebrate experimental systems, alternative splicing regulation is the result of dynamic antagonism between transacting factors binding to positive or negative elements in the pre-mRNA and promoting or repressing the use of alternative splice sites. Some of these elements bind cell-specific splicing factors. For example, neuron-specific inclusion of alternative exons in several pre-mRNAs is mediated by binding of the activator Nova-1, which is expressed only in neurons. This neuron-specific activity is antagonized by binding of the ubiquitously expressed repressor polypyrimidine tract-binding protein (PTB) in non-neuronal cells (1).The muscle-specific alternative exon 5 of cardiac troponin T (cTNT) 1 is also regulated by antagonistic activities. Musclespecific splicing enhancers (MSEs) in cTNT pre-mRNAs regulate inclusion of exon 5 in striated muscle by binding to activators and repressors of splicing. Negative elements within MSEs upstream and downstream of exon 5 repress exon inclusion in non-muscle cells by binding PTB (2).

Cardiac Tissue-Specific Repression of CELF Activity Disrupts Alternative Splicing and Causes Cardiomyopathy

“…Another possible explanation for the incomplete restoration of wild-type phenotype in the double-transgene animals is that we are only adding back a single CELF protein. ETR-3 is also expressed in the heart (19,21,22) and may exert some effects on splicing that do not overlap with CUG-BP1. Although CUG-BP1 and ETR-3 are highly conserved (the human proteins are 78% identical), they may interact with different subsets of transcripts or different protein partners to form distinct splicing complexes on their target RNAs.…”

“…Members of the CUG binding protein (CUG-BP) and the embryonic lethal abnormal vision type RNA binding protein 3 (ETR-3)-like factor (CELF) family of RNA binding proteins (also called BRUNOL proteins) regulate alternative splicing by binding to intronic elements within specific pre-mRNA targets (7,10,19,21,35,38). Two CELF proteins, CUG-BP1 (also known as BRUNOL2) and ETR-3 (also known as BRUNOL3, CUG-BP2, or NAPOR), are expressed in the heart and are hypothesized to drive changes in alternative splicing during cardiac development (22).…”

“…It is currently estimated that 60 to 74% of human genes undergo alternative splicing (17,18). Tight regulation of alternative splicing is necessary for the appropriate temporal and spatial control of gene expression, and disruption of splicing regulation can cause or contribute to human disease (11).Members of the CUG binding protein (CUG-BP) and the embryonic lethal abnormal vision type RNA binding protein 3 (ETR-3)-like factor (CELF) family of RNA binding proteins (also called BRUNOL proteins) regulate alternative splicing by binding to intronic elements within specific pre-mRNA targets (7,10,19,21,35,38). Two CELF proteins, CUG-BP1 (also known as BRUNOL2) and ETR-3 (also known as BRUNOL3, CUG-BP2, or NAPOR), are expressed in the heart and are hypothesized to drive changes in alternative splicing during cardiac development (22).…”

Exon Selection in α-Tropomyosin mRNA Is Regulated by the Antagonistic Action of RBM4 and PTB

“…Moreover, various splicing regulatory proteins bind to intronic splicing enhancers. For example, CELF family members, such as CUG-BP and ETR-3 proteins, are involved in a number of muscle-or neuron-specific splicing regulation events via CUG-or UG-rich elements (5,17,19). Neuronspecific Nova proteins activate the exon inclusion of several pre-mRNAs encoding neurotransmitter receptors by binding to adjacent intronic UCAU repeated sequences (9,25).…”

Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays

“…It is believed that the ability of the auxiliary cis-elements to regulate splicing is generally mediated by RNA-binding proteins that interact with them and either promote or hinder splicing at adjacent splice sites. Such splicing regulatory proteins include ubiquitously expressed factors as well as those with a more tissue or celltype limited pattern of expression (Bourgeois et al 2004;Ladd et al 2004;Ule et al 2005;Underwood et al 2005). The best characterized elements have been the ESEs and ESSs, which have been shown to play a role in splicing of both constitutive as well as alternatively spliced exons (Zheng 2004).…”