Intracellular localisation of SF1

Collectively, the results presented in this part of the study have shown that SF1 is diffusely distributed in the nucleoplasm, excluded from nucleoli and concentrates, like other splicing factors, in speckles, but also in paraspeckles, whose function in not well understood. This localisation of SF1 is sensitive to the transcriptional state of the cell and the protein is redistributed after ActD treatment to perinucleolar caps and enlarged speckles, and, in addition, is found in foci with SMN.

SF1 localisation in speckles

In mammalian cells splicing factors localise in the nucleoplasm at sites of transcription and are also found concentrated in speckles (Lamond and Spector, 2003). In addition, it has recently been reported that SF1 together with the U2AF heterodimer localises in splicing speckles, when it is not involved in splicing, due to an excess of RNA molecules at these sites (Rino et al., 2008). The presence of SF1 in speckles was thus not surprising. Furthermore, nuclear speckles are highly dynamic compartments.

When transcription is active, splicing factors are recruited from speckles and translocate to sites of gene activation (Misteli et al., 1997). Conversely, when transcription is off, they concentrate in speckles, which consequently appear enlarged (Misteli et al., 1997). From our results it is evident that SF1 follows the same pathway as other splicing factors after inhibition of transcription. Whether only diffused nucleoplasmic SF1 concentrates in speckles after transcriptional arrest or, in addition, the portion of the protein that is present in paraspeckles, remains to be clarified.

SF1 as a new paraspeckle component

The included manuscript contains a discussion both for the localisation of SF1 to paraspeckles as well as for its translocation to perinucleolar caps after transcription inhibition. Therefore, here I will only discuss the significance of RNA for SF1 in the context of paraspeckles.

Paraspeckle integrity depends on RNA (Clemson et al., 2009; Prasanth et al., 2005;

Sasaki et al., 2009; Sunwoo et al., 2009). However, RNA is not important for the localisation of paraspeckle components at the nucleolar periphery (Clemson et al.,

2009; Fox et al., 2005; Sasaki et al., 2009; Sunwoo et al., 2009). In agreement with this, the data presented in the included monuscript show that RNA is also necessary for the localisation of SF1 to paraspeckles, but not to perinucleolar caps. However, results from the truncation mutants show that the RNA binding domain of SF1 is dispensable for its paraspeckle localisation and that the targeting signal(s) resides in the C-terminal part of the protein. Together these results imply that although the structural core of paraspeckles is RNA, SF1 is targeted to and kept in these structures via a binding partner, which remains to be identified.

We have further demonstrated that SF1 is in the same complex with other paraspeckle proteins, both in transcriptionally active and inactive cells. However, in contrast to other paraspeckle components that appear to associate with each other through protein-protein interactions (Fox et al., 2005; Myojin et al., 2004; Peng et al., 2002), SF1 appears to be linked to PSP1, p54^nrb and CFIm68 through RNA, since anti-SF1 antibodies do not co-immunoprecipitate other paraspeckle components from HeLa cell lysates treated with RNase. These data suggest that in paraspeckles SF1 may have a role distinct from that of other components of these structures.

SF1 colocalisation with SMN

In a study to examine the dynamic reorganization of speckles after transcription inhibition, Wang and co-workers (Wang et al., 2006) discovered that enlarged speckles contained a ‘hole’, which was evident after staining with the protein-marker for speckles, SC35. These SC35-free holes were found to be filled with phosphorylated RNA pol II and SMN. In addition, the holes appeared to maintain low levels of transcriptional activity. It was therefore proposed that the reorganized speckles are sites of residual transcription after drug treatment and prominent transcription after drug removal.

As mentioned in the manuscript, in transcriptionally active cells SF1 is not present in gems. However, after ActD treatment, we found SF1 with SMN in structures surrounded by SF2/ASF that correspond to the hollow holes identified by Wang et al.

(Wang et al., 2006). Interestingly, the same structures were also occupied by the U2AF heterodimer. Although a role for SF1 in transcription has been reported, U2AF has only been associated with maintenance of chromatin and nuclear structure in S.

pombe (Beales et al., 2000). Given that SMN is involved in the assembly of snRNPs (Pellizzoni et al., 2002), and since both SF1 and U2AF are involved in splicing, these

structures may, in addition to transcription, be involved in the processing of pre-mRNAs that are transcribed in them.

In summary, all SF1 isoforms tested in this study show a similar distribution under the conditions tested, which we consider as an indication that they all function in similar pathways. The experiments for the different SF1 isoforms were performed with transiently transfected cells that overexpressed SF1. Future experiments should focus on investigating the dynamics of different SF1 isoforms using stable cell lines and live imaging. Using live microscopy, it would be interesting to clarify whether there are two distinct pools of SF1, one in speckles and one in paraspeckles, and if these pools behave differently under different conditions, such as transcription inhibition and stress. If this were the case, it would clearly indicate at least two distinct functions for SF1.

Paraspeckles are probably not sites of splicing

All the so far known components of paraspeckles are reported to be involved in mRNA processing and metabolism. Localisation of splicing factors SF1 and U2AF in these structures therefore could point to a role for paraspeckles in splicing. SF3a is a constitutive splicing factor shown both in vivo and in vitro to be essential for splicing (Tanackovic and Krämer, 2005). Here we have shown that the three subunits of SF3a (120, 60, and 66) are absent from paraspeckles. In addition, the phosphorylated form of SF3b155, which is a subunit of the constitutive splicing factor SF3b (Brosi et al., 1993b), was also absent from these structures. Our results therefore argue against a role for paraspeckles in splicing and suggest a function for SF1 and U2AF in these structures different from splicing. Intriguingly, SF1 has been implicated in a number of processes detailed in the introduction of this thesis. In addition, U2AF⁶⁵ has been shown to associate with a subset of spliced mRNAs in human cells (Gama-Carvalho et al., 2006), to be required for the export of several intronless mRNAs in Drosophila (Blanchette et al., 2004) and is also implicated in cell cycle progression and maintenance of chromatin and nuclear structure in S. pombe (Beales et al., 2000). The elucidation of the role of paraspeckles in the cell would clearly shed light on the function of both SF1 and U2AF in these structures.

SF1 in stress

CTN-RNA is a paraspeckle-retained polyadenylated RNA that regulates the expression of its protein-coding partner mCAT2 (Prasanth et al., 2005). It is released from paraspeckles under stress conditions such as viral infection, and is cleaved to produce an mRNA containing the mCAT2-coding region, thus contributing to a rapid response of the cell to stress. Consistent with a role of paraspeckles in the stress response, in this study we have shown that TIA-1, a protein-marker for SGs is also present in paraspeckles. We have further shown that a portion of SF1 translocates to SGs upon induction of stress. It will be interesting to identify the origins of this SF1 population and clarify whether it is the paraspeckle pool of SF1 that translocates to the SGs. In addition, preliminary data from our lab show that another two proteins found in SGs are also present in paraspeckles. In contrast, under the same stress conditions, p54^nrb, although displaced from paraspeckles, does not translocate to the cytoplasm.

The fate of other known paraspeckle components, such as PSP1 and CFIm68, needs to be investigated.

Finally, although a protein-coding partner like in the case of CTN-RNA has not been identified for the ncRNAs Malat-1 and Men-ε/β detected in paraspeckles, it would be interesting to investigate the localisation of these ncRNAs under stress conditions.

Given its shuttling ability, SF1 might be involved in exporting some of these ncRNAs to the cytoplasm under stress conditions. The localisation of SF1 in SGs and the link with paraspeckles clearly needs further investigation.