SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements

Abstract

Interphase chromatin is arranged into topologically separated domains comprising gene expression and replication units through genomic sequence elements, so-called MAR or SAR regions (for matrix- or scaffold-associating regions). S/MAR regions are located near the boundaries of actively transcribed genes and were shown to influence their activity. We show that scaffold attachment factor B (SAF-B), which specifically binds to S/MAR regions, interacts with RNA polymerase II (RNA pol II) and a subset of serine-/arginine-rich RNA processing factors (SR proteins). SAF-B localized to the nucleus in a speckled pattern that coincided with the distribution of the SR protein SC35. Furthermore, we show that overexpressed SAF-B induced an increase of the 10S splice product using an E1A reporter gene and repressed the activity of an S/MAR flanked CAT reporter gene construct in vivo . This indicates an association of SAF-B with SR proteins and components of the transcription machinery. Our results describe the coupling of a chromatin organizing S/MAR element with transcription and pre-mRNA processing components and we propose that SAF-B serves as a molecular base to assemble a 'transcriptosome complex' in the vicinity of actively transcribed genes.

Stress-induced Nuclear Bodies Are Sites of Accumulation of Pre-mRNA Processing Factors

“…Both splicing factors were expressed as GFP fusions in transiently transfected HeLa cells and their association with stress-induced SNBs was verified by confocal laser microscopy on cells stained with the anti-HAP antibody. As exemplified in Figure 6, SRp30c was recruited to the bodies as efficiently as HAP, suggesting that the two factors not only physically interacted with each other (Nayler et al, 1998) but also participated to a common metabolic pathway that entailed their redistribution after stress treatments. In regard to 9G8, the recruitment to stress-induced SNBs, albeit clearly detectable, occurred only in a fraction (ϳ30%) of the trans- fected cells.…”

“…In addition, we selected two clones containing the entire ORF for 9G8, another member of the family of SR splicing factors, and a few clones for as yet uncharacterized proteins. The interaction, both in vitro and in vivo, between HAP and SR splicing factors, particularly SRp30c, is not a novelty (Nayler et al, 1998). However, it is intriguing that these splicing factors can bind to the region necessary for the recruitment of HAP to stress-induced SNBs as if the interaction with SRp30c and association of HAP with the bodies after heat shock were intrinsically connected.…”

“…First, HAP contains a canonical RNA binding domain and is part of the hnRNP complexes (Weighardt et al, 1999). Second, in a two-hybrid assay HAP binds to hnRNP A1 (Weighardt et al, 1999) and to the C-terminal domain of RNA polymerase II (Nayler et al, 1998). In addition, HAP interacts, both in vitro and in vivo, with a number of splicing factors, among which are SRp30c, SF2/ASF, and htra2-beta (Nayler et al, 1998), and with Sam68 and SLM-2 (Stoss et al, 2001).…”

“…Second, in a two-hybrid assay HAP binds to hnRNP A1 (Weighardt et al, 1999) and to the C-terminal domain of RNA polymerase II (Nayler et al, 1998). In addition, HAP interacts, both in vitro and in vivo, with a number of splicing factors, among which are SRp30c, SF2/ASF, and htra2-beta (Nayler et al, 1998), and with Sam68 and SLM-2 (Stoss et al, 2001). Finally, a direct involvement of HAP in splicing is suggested by the observation that its overexpression affects splicing of the adenovirus E1A reporter gene (Nayler et al, 1998).…”

Novel role of the RET finger protein in estrogen receptor-mediated transcription in MCF-7 cells

“…SAFB1/2 are nuclear matrix proteins characterized by an N-terminal DNA-binding domain (SAF-box) that binds to scaffold/matrix attachment regions (S/MARs), and a central RNA recognition motif (RRM) [10]. SAFB1 interacts with the RNA processing machinery and RNA polymerase II, suggesting that it is part of a "transcriptosome" complex coupling chromatin structure to transcription and RNA processing [11]. We have also shown that the C-terminus of SAFB1/2 harbors a strong independent repression domain that can mediate repression when transferred to a heterologous DNA-binding protein.…”