A brain-derived MeCP2 complex supports a role for MeCP2 in RNA processing

MeCP2 was originally identified by its ability to preferentially bind double stranded DNA containing symmetrically methylated CpG dinucleotides and is the founding member of the methyl-CpG binding domain (MBD) family of proteins [1,2]. The first biological role for MeCP2 was illustrated by showing the protein interacts with methylated DNA in vivo and could repress transcription by association with a transcriptional co-repressor complex containing Sin3A and histone deacetylase [3–5]. In 1999 a genetic analysis identified mutations in MECP2 as causal for Rett Syndrome (RTT), providing the first direct link between an epigenetic regulator and a human disease [6]. RTT is a severe postnatal neurodevelopmental disorder and one of the most common causes of mental retardation in females [7]. First described in 1966 by Andreas Rett [8], RTT is characterized by a period of apparently normal development from birth to 6–18 months followed by a regression of obtained language and motor skills [7]. RTT patients usually exhibit a deceleration of head growth, respiratory dysfunction, scoliosis, cognitive impairment, seizures, and social withdraw [8,9]. In addition to RTT, numerous MECP2 mutations have now been linked to a variety of additional disorders, including autism, Angelman syndrome, learning disabilities, and mental retardation syndromes [7,10–14]. MeCP2 has been reported to associate with myriad protein partners including Sin3A [3,5], c-REST and Suv39h1 [15], c-Ski and N-CoR [16], Brm [17], and HP1 [18], all supporting a model of MeCP2 interacting with or being a stable component of transcriptional co-repressor complexes, resulting in targeted transcriptional repression of methylated DNA through modification of the chromatin state or chromatin associated proteins. However, the biological relevance and implications towards RTT for these numerous documented MeCP2 interactions is not clear due in part to the particular methods utilized and non-neuronal choices for initial cellular protein sources. In fact, contradicting these numerous studies, it has been proposed that endogenous MeCP2 does not form any stable protein-protein interactions in vivo [19]. Compounding the issue, recent work has expanded MePC2’s proposed gene regulatory role beyond mere transcriptional repression; MeCP2 is implicated in transcriptional activation, genome-wide transcriptional silencing, mediating chromatin and nuclear architecture, and regulating pre-mRNA splicing as well [20–23]. Thus, the in vivo protein-protein interaction profile of endogenous MeCP2, particularly in the brain, is still an open question and increasingly more important to understand as new functions for MeCP2 are emerging. Genetic studies in mice suggest that expression of functional MeCP2 in neurons is essential for normal synapse formation and neuronal function during postnatal development and re-expression of MeCP2 in differentiated neurons alone rescues a RTT mouse model [24–29]. However, this idea is being challenged by a recent study that indicates the lack of MeCP2 specifically in glial cells contributes to RTT phenotypic neurons by an unknown secreted glial factor [30]. This discrepancy illustrates the need for more unbiased approaches in determining the molecular roles of MeCP2 in both normal and RTT brain; thus, intact mammalian brain tissue would be the ideal source to study endogenous MeCP2 protein function. Here we use the power of biochemistry to characterize MeCP2 in the mammalian brain and show that native MeCP2 protein purified from adult rat brain exists in multiple biochemically distinct pools/complexes, consistent with MeCP2 working as a multi-functional protein. We further characterize one brain-derived MeCP2-complex that contains Prpf3, a known spliceosome-associated protein [31], as well as the Sdccag1 [32], a mediator of nuclear export [33]. MeCP2 shows specific, direct interactions with Prpf3 and Sdccag1 and these interactions are disrupted by certain RTT mutations. In addition, we show that MeCP2 and Prpf3 co-associate in vivo with mRNAs from genes activated by MeCP2, further supporting the previously identified regulatory role of MeCP2 in mRNA biogenesis [23] and providing another potential mechanism disrupted during RTT pathogenesis.