Findings presented here further our understanding of nuclear structure and mRNA transport and, at the same time, provide insight into the cellular pathogenesis of DM1. We show that newly synthesized DMPK RNA accumulates within the interior of an SC-35–defined domain in normal muscle nuclei, having emanated from a gene positioned at the domain's edge. In contrast, the mutant DMPK transcripts in DMI detach from the gene but are not within the domain; rather, they accumulate in multiple granules that gather at the edge of domains. This change in domain association occurs in concert with mutant DMPK RNA retention in the nucleus. Although the transcripts do not appear to enter the SC-35 domain, they are nonetheless spliced. Down-regulation of MBNL1 changes the distribution of newly transcribed mutant DMPK RNA such that it is now found within the SC-35 domain.
Our studies examine the detailed relative distributions of gene/RNA/protein associated with normal versus mutant alleles but in static images that capture a window of time. A major advantage of this approach is that it provides information about the real endogenous gene and RNA, which are expressed in a native structural as well as physiological context. Although this does not provide direct visualization of molecules in live cells, our findings collectively provide evidence of the route occupied by at least a substantial fraction of these native transcripts. Because transcripts clearly emanate from the gene, for the sake of this Discussion, we consider the gene point A and will consider any accumulation of transcripts at a resolvable distance from the corresponding gene to have moved with respect to it.
Collectively, these findings suggest several important points regarding the relationship of the SC-35 domains to mRNA metabolism and transport. (1) The consistent positioning of both homologous DMPK
genes at the outer edge of an SC-35 domain adds to the body of evidence that specific active genes are organized relative to SC-35 domains, providing an example of an associated gene that is not particularly complex or highly expressed. (2) We interpret the accumulation of transcripts within the SC-35 domain (to one side of the gene at the domain edge) to indicate a normal step in the path of DMPK
mRNA for at least a substantial fraction of DMPK
transcripts. The fact that mutant transcripts do not enter these domains further supports the idea that entry into the domains is a step that can be blocked by mutation. (3) Because MRGs contain spliced mRNA, passage into the splicing factor–rich domain does not appear to be required for splicing, which is consistent with other evidence that most splicing occurs at the domain periphery (Johnson et al., 2000
). These findings are consistent with the idea that postsplicing steps linked to export may occur within the domain. (4) Blockage of mutant transcripts before entering SC-35 domains provides further evidence for the structural integrity of the SC-35 domain (with which normal and mutant alleles remain associated), which exists independently of the presence of DMPK
mRNA within it. Consistent with this, the normal DMPK
mRNA does not occupy the whole domain of splicing factors, as might be expected if the domain was merely factors bound to this pre-mRNA. (5) Loss of DMPK
RNA signals upon transcription inhibition demonstrates that the normal DMPK
RNA accumulation in SC-35 domains does not represent RNA just trapped within the domain but is chased as would be expected for a transported mRNA. (6) Remarkably, of the two mutant pre- mRNAs studied thus far, both have shown abnormal accumulations at or within the SC-35 domains and not at other sites such as the nuclear envelope or nucleolus. Whereas mutant COL1A1 RNA in osteogenesis imperfecta accumulates to abnormal levels within the domain, mutant DMPK
transcripts accumulate outside the domain. This supports a model in which passage into and release from these domains are distinct steps in the normal path of some pre-mRNAs.
Although we demonstrate that many DMPK
transcripts are not randomly dispersed upon initial release from the gene, these results should not be misinterpreted as excluding the possibility that export of these mRNAs may also involve multidirectional diffusion, which is consistent with other models of RNA trafficking (Politz et al., 2003
; Gorski et al., 2006
). These collective findings support a model of early steps in mRNA export and maturation as presented in .
Figure 10. Model of SC-35 domain function. In this model, DMPK genes are positioned at the edge of SC-35 domains. The RNAs produced from these loci are primarily spliced at the edge of the domain. These mRNAs then transit into the domain (step 1). In the interior (more ...)
This study provides evidence of a relationship between DMPK
mRNA and SC-35–defined speckles that is disrupted when a CUG expansion in DM1 prevents normal mRNA export. Despite numerous studies describing the association of specific mRNAs with SC-35–defined speckles (Xing et al., 1995
; Jolly et al., 1999
; Smith et al., 1999
; Shopland et al., 2002
), the idea that SC-35 domains contain mRNAs and/or play a role in mRNA export remains somewhat controversial (Hall et al., 2006
). Therefore, we will briefly address the two major pieces of evidence that generate this uncertainty. First, the belief that SC-35 speckles do not contain appreciable short-lived pre-mRNAs is based on early findings that tritiated uridine incorporation labels the speckles (interchromatin granule clusters) very little relative to the surrounding nucleoplasm (Fakan and Bernhard, 1971
; Fakan, 1994
; for review see Spector, 1993
). However, a study did find newly synthesized BrUTP-labeled RNA concentrated within these domains (Wei et al., 1999
). Most importantly, labeling methods such as [3
H]uridine or bromo-UTP are wholly nonspecific and do not necessarily reflect the distribution of pre-mRNA. These methods primarily label unknown heterogeneous nuclear RNA (Salditt-Georgieff et al., 1981
) and introns (most of pre-mRNA mass) that are rapidly removed and disperse during the labeling period (Xing et al., 1993
; for review see Moen et al., 1995
). Evidence in support of this concern has been strengthened in recent years by findings that transcription of nongenic DNA is far more widespread throughout the genome than previously anticipated (Cheng et al., 2005
; Johnson et al., 2005
). Furthermore, copious amounts of RNA throughout the nucleoplasm are detected by RNA hybridization using Cot-1 DNA, composed largely of repetitive elements such as Alu and long interspersed nuclear elements (Hall et al., 2002
), which recent findings suggest is largely nongenic transcription (Hall et al., 2002
; Chaumeil et al., 2006
; Clemson et al., 2006
). In short, uridine incorporation detects a great deal of RNA that is not mRNA and, as such, does not accurately represent the distribution of pre-mRNA in the nucleus.
Second, the demonstration that the poly(A) RNA signal often remains in SC-35 domains upon transcription inhibition (Lawrence et al., 1993
; Huang et al., 1994
) is often interpreted to indicate that this poly(A) RNA is not mRNA but a putative long-lived, polyadenylated, structural RNA. However, transcriptional inhibition has complex effects on nuclear RNA distributions and transport that varies with the RNA. In a dramatic example of this, we recently identified an abundant noncoding polyadenylated nuclear RNA, NEAT1 (Hutchinson et al., 2007
), that actually enters the SC-35 domains upon treatment with certain transcription inhibitors (unpublished data). This illustrates the difficulty of interpreting the nature of the poly(A) RNA in domains from transcription inhibition experiments. Our study demonstrates that at least some specific mRNAs, such as DMPK
RNA, do leave the SC-35 domain upon transcription inhibition. In addition, recent studies of labeled RNAs introduced into live cells indicate that poly(A) RNA (Molenaar et al., 2004
) and specific mRNAs (Tokunaga et al., 2006
) passage through domains and that export-ready mRNA is present in speckles (Schmidt et al., 2006
). Thus, although our study takes a different approach to study naturally occurring mutations of an endogenous RNA, our findings complement and substantially extend approaches that seek to understand the behavior of endogenous molecules by examining labeled, microinjected RNAs in live cells.
In addition to its relevance for fundamental nuclear structure, this study contributes insight into the cellular pathogenesis of DM1. (1) We have identified a point in nuclear structure at which the paths of normal and mutant DMPK mRNA diverge. (2) As this difference correlates with cytoplasmic mRNA export, it defines the intranuclear step at which the block in transport of many or all mutant DMPK transcripts likely occurs. (3) We provide evidence that the formation of MRGs mediated by MBNL1 is responsible for blocking the mutant RNA from entry into the domain. Our analysis indicates that depletion of newly synthesized MBNL1 allows newly transcribed DMPK RNA carrying CUG repeat expansions to accumulate within SC-35 domains in a manner similar to that seen with normal DMPK RNA. MBNL1 may act in concert with other factors, such as hnRNP H, to bind CUG repeat RNA and form the RNA/protein aggregates. Thus, both the nuclear retention of mutant DMPK RNA as well as sequestration of specific nuclear factors like MBNL1 and hnRNP H are likely involved in the complex DM1 phenotype.