Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific

Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript trees of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional tracks of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism

Unbalanced X;autosome translocations provide evidence for sequence specificity in the association of XIST RNA with chromatin

Whether XIST RNA is indifferent to the sequence content of the chromosome is fundamental to understanding its mechanism of chromosomal inactivation. Transgenic Xist RNA appears to associate with and inactivate an entire autosome. However, the behavior of XIST RNA on naturally occurring human X;autosome translocations has not been thoroughly investigated. Here, the relationship of human XIST RNA to autosomal chromatin is investigated in cells from two patients carrying X;autosome translocations in the context of almost complete trisomy for the involved autosome. Since trisomies of either 14 or 9 are lethal in early development, the lack of serious phenotypic consequences of the trisomy demonstrates that the translocated autosomes had been inactivated. Surprisingly, our analyses show that in primary fibroblasts from adult patients, XIST RNA does not associate with most of the involved autosome even though the bulk of it exhibits other hallmarks of inactivation beyond the region associated with XIST RNA. While results show that XIST RNA can associate with human autosomal chromatin to some degree, several observations indicate that this interaction may be unstable, with progressive loss over time. Thus, even where autosomal inactivation is selected for rather than against, there is a fundamental difference in the affinity of XIST RNA for autosomal versus X chromatin. Based on these results we propose that even autosomal chromatin that had been inactivated earlier in development may undergo a stepwise loss of inactivation hallmarks, beginning with XIST RNA. Hence compromised interaction with XIST RNA may be a primary cause of incomplete or unstable autosomal inactivation