We have identified RHA as a novel SMN-interacting protein and showed that this interaction is defective in SMN mutants found in some SMA patients. Coimmunoprecipitation and pull-down experiments demonstrated the physical association of the SMN complex with pol II, snRNPs, and RHA in vivo and in vitro. Importantly, the SMN complex binds pol II CTD, and RHA at least in part mediates this interaction. Thus, RHA likely plays a role in the association between the SMN complex and pol II possibly similar, but not mutually exclusive, to its role in bridging CBP and BRCA1 to pol II (Nakajima et al. 1997
; Anderson et al. 1998
). We further demonstrated that expression of SMNΔN27 brings about a profound rearrangement of pol II, RHA, snRNPs, TBP and polyadenylation factors, all of which specifically accumulate in enlarged G/CBs. This reorganization is accompanied by the inhibition of both pol I and pol II transcription, as detected in vivo by the BrU labeling method we have developed. These findings indicate a physical as well as a functional interaction between the SMN complex and components of the pol II transcription/processing machinery. Previous studies have indicated the presence of additional proteins associated with the SMN complex and the possibility that multiple SMN complexes may exist in vivo (Liu et al. 1997
; Charroux et al. 1999
, Charroux et al. 2000
; Meister et al. 2000
). The observations that RHA does not localize to gems and is not found in all SMN complexes suggest that RHA is not a core component of the SMN complex such as Gemin2, Gemin3, and Gemin4. Similar to Sm proteins of spliceosomal snRNPs, we consider RHA as a possible substrate of SMN complex function.
Our findings are consistent with the possibility that the assembly of transcriptosomes is a process that is extrinsic to, and precedes, binding of the polymerase complex to gene promoter elements. Most importantly, we propose that the SMN complex plays a central role in the pathway of transcriptosome assembly. The physical association of the SMN complex with components of the pol II transcription/processing machinery in untransfected cells indicates that they interact under normal conditions, and expression of SMNΔN27 likely blocks this pathway. Although no dominant–negative mutations have been reported in SMA patients, possibly because they would result in embryonic lethality, a point mutation in exon 1 has been identified in three unrelated SMA patients (Parsons et al. 1998
). This suggests that the amino acid sequence encoded by exon 1, which is deleted in SMNΔN27, is important for SMN function and SMA. Moreover, we recently identified a dominant-negative mutant of Gemin2 that displays a phenotype similar to SMNΔN27 (Pellizzoni, L., and G. Dreyfuss, manuscript in preparation). The observation that expression of mutants of two different proteins of the SMN complex leads to the dramatic reorganization of components of the pol II transcription/processing machinery, and the inhibition of transcription in vivo argues that these effects result from disruption of a genuine pathway that requires SMN functions in normal cells.
Pol II CTD undergoes cycles of phosphorylation and dephosphorylation that modulate its association with processing factors and its activity in transcription (Dhamus 1996
). Pol IIa binds to the preinitiation complex on promoter elements, and CTD phosphorylation is believed to trigger release from the promoter and initiation of transcription (Lu et al. 1991
; Akoulitchev et al. 1995
). Pol IIo drives elongation and pre-mRNA processing events and is released from the gene upon termination of transcription (O'Brien et al. 1994
). After their release from sites of transcription, pol II and several components of the transcription and pre-mRNA processing machineries seem to follow a common pathway in the nucleus (Zeng et al. 1997
). A pool of both pol II isoforms exists in the nucleoplasm that is not engaged in active transcription and may represent the fraction of pol II undergoing recycling or regeneration between rounds of transcription (Kim et al. 1997
; Zeng et al. 1997
). Dephosphorylation of pol II CTD is one of the steps required for pol II recycling (Cho et al. 1999
). The observation that the SMN complex associates with both pol II isoforms but only pol IIa accumulates in the G/CBs upon SMNΔN27 expression, suggests that pol II CTD dephosphorylation may take place in association with components of the SMN complex and that SMNΔN27 blocks the pathway downstream of this event.
HnRNP and SR proteins represent indirect markers of pre-mRNA distribution in the nucleus, and these abundant RNA-binding proteins do not accumulate in G/CBs (). Moreover, hnRNP proteins do not coimmunoprecipitate with the SMN complex (; data not shown). These results suggest that the SMN complex associates with transcriptosome components that are not bound to pre-mRNAs. It is tempting to speculate that pol II and transcriptosome components associate with the SMN complex after their dissociation from transcribing genes and before being recruited for a new round of transcription.
TBP is a general transcription factor common to all three RNA polymerases (Hernandez 1993
; Rigby 1993
). TBP accumulates in the G/CBs upon expression of SMNΔN27, and likely as a consequence pol I transcription in the nucleolus is strongly inhibited. Similar to the case of pol II, RNA polymerase I holoenzyme as a transcriptionally competent large multisubunit complex has been identified, and the possibility that it may be preassembled in CBs has been suggested (Seither et al. 1998
; Gall et al. 1999
). By extension of what we have observed here for components of pol II transcriptosomes, the SMN complex may interact with components of the pol I transcription machinery and also play a role in the assembly of a class I transcriptosome. Experiments are underway to assess this possibility.
Our findings suggest a critical role for the SMN complex in several aspects of mRNA biogenesis. Neurons are likely to be particularly sensitive to defects in mRNA metabolism as neuronal differentiation and maintenance require highly active and finely regulated pre-mRNA transcription and processing activities (Tanabe and Jessell 1996
; Grabowski 1998
). It is conceivable that the drastic reduction in the amount of SMN in motor neurons of SMA patients leads to an impaired capacity to produce mRNAs, and thus leads to neuronal degeneration.