Additionally, transfection of either GABRGi5ID or GRIK1i1ID had n

Additionally, transfection of either GABRGi5ID or GRIK1i1ID had no effect on either FMR1i1 selleck chemicals or CAMK2Bi3 localization (Figure S4), confirming the specificity of the FMR1i1ID1- and CAMK2Bi3ID1-driven mechanisms. To assess whether transcript localization affects the location of the encoded protein product, we

visualized the subcellular distribution of FMRP, which is encoded by FMR1, by using immunofluorescence. FMRP is normally distributed throughout both the cell soma and dendrites of neurons, as was the case for cells transfected with EGFP (Figure 4A). Upon transfection with FMR1i1ID1-EGFP, the relative amount of FMRP in the dendrites decreases, with FMRP concentrating at the outer boundaries of the soma (Figure 4A). In contrast, subcellular distribution of CAMK2B protein is unaffected by FMR1i1ID1-EGFP transfection (Figure 4B). Thus, the function of this ID element containing CIRT is consistent with a role in regulating dendritically localized FMRP protein levels and subsequent function in the dendrite. By using three independent methods of detection in multiple cell cultures, we have described a large number of previously unreported intronic sequences in the dendritically localized mRNA of primary rat hippocampal neurons. These CIRTs represent a class of transcript that has important cellular function in neurons including involvement in dendritic targeting of mRNAs via co-opted

retrotransposons. These dendritic targeting elements are additionally notable in that they occur outside of the transcript UTR or coding region and are found in more than two different gene transcripts. Our BVD-523 concentration in situ hybridization results show a variety of dendritic distribution patterns, suggesting that localization is a complex process that likely involves multiple ID element-dependent and -independent mechanisms. The fact that exogenous expression of any particular intronic ID element does not necessarily disrupt targeting of all intron-retaining transcripts suggests the existence isothipendyl of multiple variant targeting mechanisms—if

only a single mechanism existed, transfection of any intronic ID element would block the targeting of all endogenous intron-retaining transcripts containing an ID element. Our data reflect at least three targeting mechanisms for intron-retaining transcripts in dendrites: one that is distinct for FMR1i1ID1, one that is common to CAMK2Bi3iD1 and FMR1i1ID1, and at least one that is ID element independent. Further, we observed that FMRP localization is directly affected by the disruption of endogenous ID element-mediated FMR1i1 targeting, suggesting that CIRTs may be critically important in the presumptive role of FMRP in modulation of local dendritic protein synthesis (Huber et al., 2002 and Weiler et al., 1997). BC1-like ID elements have been implicated in brain-specific gene regulation since their discovery (Milner et al.

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