Supplementary results

Cellular distribution of His-tagged SF1 isoforms

In a first step in the attempt to elucidate the localisation of individual SF1 isoforms, cDNAs encoding ten isoforms were cloned into the pcDNA3.1/His vector. The exon organization of these isoforms is shown in Figure 9. All isoforms tested for cell localisation, apart from GKS-Ala-B, have been identified in HeLa cells (Arning et al., 1996; Caslini et al., 1997; Krämer et al., 1998; Tanackovic and Krämer, 2005; Toda et al., 1994; Zhang and Childs, 1998; Tanackovic et al., unpublished data).

Figure 9. Schematic representation of the SF1 isoforms analysed. In total, ten SF1 isoforms were cloned into different vectors and their cellular localisation was analysed by fluorescence microscopy or immunofluorescence. Of these isoforms, only the existence of GKS-Ala-B has not yet been verified (marked with *). Since the 5’ half of the isoforms is common, the scheme only shows the exon structures beyond exon 9. Boxes represent exons, different colours depict different reading frames, and the stars show the location of stop codons.

To verify proper expression and correct size of the isoforms, 293T cells were transiently transfected with the plasmids and whole cell extracts were prepared 48 hr post-transfection. All His-tagged SF1 isoforms were abundantly expressed and migrated in the SDS gel with expected sizes (Figure 10).

Figure 10. Western blot of His6-tagged SF1 isoforms. 293T cells were transiently transfected with pcDNA/His plasmids carrying individual SF1 isoforms. Cells were collected 48 hr post-transfection and total cell lysates were prepared and separated on a 7.5% SDS gel.

Western blotting with anti-His revealed that all isoforms were successfully expressed, with the size of the migration corresponding to the calculated size for individual isoforms.

Subsequently, HeLa cells were transiently transfected with individual isoforms and stained with an anti-His antibody. pcDNA3.1/His vector was also transfected as a control. Cells were examined by immunofluorescence, and representative images for each construct together with the negative control are shown in Figure 11A and B, respectively. All SF1 isoforms were diffusely distributed in the nucleus, excluded from nucleoli and, with the exception of the short D-type isoforms, clearly concentrated in distinct foci corresponding to paraspeckles. All isoforms (with the exception of Ala-A) were subsequently subcloned into pEGFP (and in some cases also into pHcRed) vector to further study their localisation. IF data for seven of the GFP-tagged isoforms is shown in the manuscript.

Figure 11. SF1 isoforms show a similar nuclear distribution. HeLa cells grown on coverslips were transiently transfected with pcDNA-SF1 isoforms or pcDNA vector alone, and collected 48 hr post-transfection for immunofluorescence. Cells were fixed and stained with anti-His to detect over-expressed His-tagged SF1 isoforms. (A) Transfections with pcDNA-SF1 isoform plasmids.

His-tagged SF1 isoforms are diffusedly distributed in the nucleoplasm, excluded from the nucleoli and concentrate in punctate structures in the nucleoplasm. For isoforms of type D, this punctate distribution is not very obvious. (B) Cells transfected with the pcDNA vector only were used as control. Only background staining with anti-His was observed in this case. The paraspeckle fixation method was used.

SF1 localises to paraspeckles but not to gems, CBs or PML bodies

As is described in the manuscript, to identify the distinct bright structures showing a concentration of SF1, HeLa cells transiently transfected with GFP-SF1 isoforms were stained with antibodies to marker proteins for selected sub-nuclear compartments.

Cells were stained with anti-PSP1, anti-SMN, anti-p80 coilin and anti-PML, as markers for paraspeckles, gems, CBs and PML bodies, respectively. The results shown in Figure 12 indicate colocalisation of SF1 with PSP1 in paraspeckles. SF1 was not present in any of the other structures examined.

Figure 12. SF1 colocalises with PSP1 in paraspeckles, but is not found in other nuclear bodies. HeLa cells were transiently transfected with GFP-GKS-Ala-B and coverslips were collected 48 hr post-transfection for fluorescence microscopy. Counter-staining with anti-PSP1, SMN, p80 and PML antibodies (as indicated) revealed that SF1 localises in paraspeckles and is not present in gems, CBs or PML bodies. The paraspeckle fixation method was used.

The short D-type isoforms of SF1 also localise in paraspeckles

Most of the SF1 isoforms examined showed a clear association with paraspeckles.

However, this was not the case for the three shortest isoforms, Pro-D, GKS-Ala-D and Ala-D, tagged with either His6 or GFP. These proteins were more diffusely distributed in the nucleoplasm than the larger isoforms and only very weak staining of small foci was evident. To verify that the faint structures observed corresponded to paraspeckles, GFP-GKS-Ala-D was transiently expressed in HeLa cells followed by staining with an anti-PSP1 antibody (Figure 13). As shown in the figure, the GFP-GKS-Ala-D isoform colocalised with PSP1 in paraspeckles. Whether the shorter isoforms of SF1 are less concentrated in paraspeckles or have a stronger nucleoplasmic concentration than the longer isoforms that makes their localisation in paraspeckles less evident, remains to be clarified. Quantification of the relative amounts for the different SF1 isoforms in each compartment by confocal microscopy could answer this question.

Figure 13. The shorter SF1 isoforms (type D) also localise to paraspeckles. HeLa cells were transiently transfected with GFP-GKS-Ala-D. Coverslips were collected 48 hr post-transfection and stained with anti-PSP1 antibody. The two proteins colocalise in paraspeckles (arrows). The paraspeckle fixation method was used.

SF1 does not colocalise with PML bodies after ActD treatment

In the manuscript, the localisation of SF1 to perinucleolar caps in transcriptionally inactive cells has been discussed extensively. After ActD treatment PML has been shown to concentrate in small structures adjacent to or on top of the nucleolar caps formed by paraspeckle proteins (Shav-Tal et al., 2005) and SF1 (our results). Figure 14 confirms that SF1 does not localise with PML in these structures.

Figure 14. SF1 does not colocalise with PML bodies after transcription inhibition. HeLa cells were transiently transfected with pEGFP-GKS-Ala-B and coverslips were collected for fluorescence microscopy 48 hr post-transfection and after a 4-hr treatment with ActD. Staining with anti-PML showed that SF1 is not present in PML-stained structures. Arrows indicate PML structures that are not observed with GFP-GKS-Ala-B. The paraspeckle fixation method was used.

GFP-tagged Ala-C and Ala-D isoforms show a distribution similar to other SF1 isoforms

In addition to the isoforms shown in the manuscript, the intracellular distribution of GFP-tagged Ala-C and Ala-D was examined. The two constructs were transiently transfected into HeLa cells followed by treatment or not with ActD. Staining of untreated and ActD-treated cells with anti-PSP1 revealed that both isoforms are present in paraspeckles and redistribute to perinucleolar caps after transcription inhibition (Figure 15).

Figure 15. GFP-Ala-C and GFP-Ala-D show the same localisation as the other GFP-SF1 isoforms. HeLa cells were transiently transfected with pEGFP-Ala-C and pEGFP-Ala-D and coverslips were collected 48 hr post-transfection for fluorescence microscopy, without (A) or with ActD treatment (B). Staining with anti-PSP1 showed that these proteins localise in paraspeckles in non-treated cells (arrows) and in perinucleolar caps in transcriptionally inactive cells (arrowheads). The paraspeckle fixation method was used.

SF1 is present in transcriptionally active sites after ActD treatment

In transcriptionally inactive cells, splicing factors concentrate in enlarged speckles.

Wang and co-workers (Wang et al., 2006) reported that these reorganized speckles consist of outer shells containing splicing factors SC35, ERK2 and SF2/ASF, as well as actin. Hollow holes observed in these structures contain RNA Poll II (mostly phosphorylated) and SMN protein. Interestingly, these structures exhibited low levels of BrdU staining and thus it has been suggested that they are major sites of residual transcription during drug-induced transcription inhibition. In the manuscript, it is shown that after transcription inhibition SF1 translocates to round, bright foci, where it colocalises with SMN. To analyse whether these structures are the same as those identified by Wang et al. (Wang et al., 2006), ActD-treated HeLa cells transiently expressing GFP-SF1 were stained with anti-SF2/ASF (Figure 16). GFP-tagged SF1 was detected in the centre of the hollow holes formed by SF2/ASF. The relevance of the presence of SF1 in these structures remains to be established.

Figure 16. SF1 ‘fills the holes’ of SF2/ASF. HeLa cells were transiently transfected with pEGFP-GKS-Ala-B and coverslips were collected 48 hr post-transfection, after a 4-hr treatment with ActD. Staining with anti-SF2/ASF antibody showed that SF1 is present in and fills the hollow structures formed by SF2/ASF (arrows).

SF1 localises to paraspeckles in various cell lines

All experiments described so far were performed in HeLa cells. Because of the somewhat variable nature of HeLa cells and to confirm the localisation of SF1 in paraspeckles, pEGFP-SF1 was transfected into HeLa cells provided by another lab, as well as into p15 and Hek293 cells. The localisation of SF1 was examined in transcriptionally active and inactive cells after staining with anti-PSP1 antibody (Figure 17). The results indicate that, in all three cell lines, SF1 is present in paraspeckles and perinucleolar caps before and after transcription inhibition, respectively. Thus, the typical localisation of SF1 is a general characteristic and not restricted to a single cell line.

Figure 17. SF1 localises to paraspeckles and perinucleolar caps in different cell lines.

HeLa (A), p15 (B) and Hek293 (C) cells were transiently transfected with pEGFP-GKS-Ala-B and coverslips were collected 48 hr post-transfection, without or with ActD treatment. Staining with anti-PSP1 antibody showed that, in all three cell lines, SF1 localises in paraspeckles in non-treated cells and in perinucleolar caps in transcriptionally inactive cells. In the merged images Hoechst staining is also shown. Arrows and arrowheads point to paraspeckles and perinucleolar caps, respectively.

The interaction of SF1 with paraspeckle components is mediated through RNA As shown in the manuscript, anti-SF1 antibodies co-immunoprecipitated other paraspeckle proteins. In order to validate the finding that SF1 is present in one complex with these proteins, the IP was repeated with anti-p54^nrb antibodies. Western blot analysis demonstrated that all paraspeckle components examined were coprecipitated with p54^nrb, and SF1 was also present in the complex (Figure 18A). We further investigated whether binding of SF1 to the other paraspeckle components is mediated through RNA. An IP was performed with anti-p54^nrb-coated beads after treatment of the extracts with RNAse A. Although all other paraspeckle components examined remained stably bound to p54^nrb, SF1 was lost from the complex (Figure 18B).

Figure 18. SF1 immunoprecipitates with p54^nrb in an RNA-dependent manner. A total cell lysate was prepared from HeLa cells and IP was performed with anti-p54^nrb-coated beads with extract that was not treated (A) or treated (B) with RNase A. Material in the different fractions of the IPs were separated on a 7.5% SDS gel and subjected to Western blotting with antibodies against protein components of paraspeckles and SF1. All components of the paraspeckles examined immunoprecipitated with p54^nrb in an RNA-independent manner. However, SF1 was only bound to the complex when RNA was present. EXT: extract after clearing, Beads: pre-clearing fraction, IP: eluted fraction, FT: flow-through, unbound fraction.

To confirm this result, an IP was performed with RNase A-treated extract and anti-SF1-coated beads. None of the paraspeckle proteins precipitated with SF1 under these conditions (Figure 19A). To see whether binding of SF1 to the paraspeckle protein complex in the perinucleolar caps also depended on RNA, the IP was repeated with an extract from HeLa cells treated with ActD. As shown in Figure 19B, no other paraspeckle proteins were detectable in the bound fraction after RNase treatment.

These results indicate that the interaction of SF1 with paraspeckle components is dependent on RNA in both transcriptionally active and inactive cells.

Figure 19. SF1 binding to paraspeckle proteins is mediated by RNA. Total cell lysates were prepared from untreated (A) or ActD-treated (B) HeLa cells. The lysates were incubated with RNase A. IPs were performed with anti-SF1-coated beads. The different fractions of the IPs were separated on a 7.5% SDS gel and subjected to Western blotting with antibodies against protein components of paraspeckles. None of the paraspeckle components examined immunoprecipitated with SF1 in either untreated or ActD-treated cells, although SF1 was successfully immunoprecipitated. EXT: extract after pre-clearing, Beads: pre-clearing fraction, IP: eluted fraction, FT: flow-through, unbound fraction.

Sequences in SF1 required for targeting to paraspeckles and perinucleolar caps All paraspeckle components identified so far contain RNA binding domains. PSP1 and CFIm68 are targeted to paraspeckles via their RNA recognition motifs (RRM) (Cardinale et al., 2007; Fox et al., 2005). In addition, the RRM of PSF has been proposed to be responsible for its localisation in these structures (Dye and Patton, 2001; Fox et al., 2005). Interestingly, it has also been shown that the domains of PSP1 required for paraspeckle and perinucleolar cap localisation are distinct, and that the RRM of the protein is dispensable for perinucleolar cap targeting (Fox et al., 2005).

In order to map the domain(s) of SF1 involved in paraspeckle and perinucleolar cap targeting and to analyse whether SF1 localisation in these structures depends on its RNA binding property, several GFP-tagged mutants comprising different regions of SF1 were constructed (Figure 20A). For mutants C7 to C18 (except C16), which lack the NLS of SF1, the NLS of the U2 snRNP protein SF3a120 was cloned between the N-terminal GFP tag and the SF1 coding sequences, to ensure nuclear targeting.

Construct C13 also contains the sequence of pyruvate kinase, a cytoplasmic protein.

All constructs were transfected into HeLa cells, whole cell lysates were prepared and proteins were separated on a 7.5% SDS gel to test for correct expression by Western blotting with anti-GFP antibodies (Figure 20B). Subsequently, the proteins were transiently expressed in HeLa cells and their localisation was examined. The table in Figure 20C summarizes the localisation of the mutant proteins. Representative images of the localisation of each SF1 mutant in transcriptionally active and inactive cells are shown in Figure 20D.

The pEGFP-NLS construct was used as control. Although the GFP-NLS protein alone was partially cytoplasmic, it was not associated with any apparent nuclear structures (Figure 20D). For mutants GFP-C11 and GFP-C14, it was difficult to see whether the proteins localised to paraspeckles. Similarly, for mutants GFP-C6 and GFP-C8 perinucleolar cap localisation was not very obvious. We therefore performed staining with anti-PSP1 antibodies in transcriptionally active cells expressing GFP-C11 and C14 (Figure 21A), as well as in ActD-treated cells expressing C6 and GFP-C8 (Figure 21B). The results obtained confirmed our initial observations that these constructs are present in paraspeckles and translocate to perinucleolar caps after transcription inhibition.

Figure 20. GFP-tagged SF1 truncation mutants. (A) Schematic representation of the GFP-SF1 truncation mutants used in this study. Boxes represent exons, different reading frames are shown in different colours and stars indicate stop codons.

(B) HeLa cells were transiently transfected with constructs encoding GFP-tagged truncation mutants and total cell lysates were prepared 48 hr post-transfection. The lysates were separated on a 7.5% SDS gel and subjected to Western blotting with anti-GFP. All constructs were expressed and their sizes corresponded to the calculated sizes.

(C) Table summarizing the nuclear localisation of GFP-tagged SF1 mutants in untreated or ActD-treated cells. - : no localisation, + : localization

D

(D) Representative images of HeLa cells transiently expressing different GFP-truncation mutants in the presence or absence of ActD. GFP-NLS expressing cells are shown as a control. Arrows and arrowheads point to paraspeckles and perinucleolar caps, respectively, in images in which the structures are present but more difficult to see.

Figure 21. Confirmation of paraspeckle and perinucleolar cap localisation of SF1 mutants C11, C14, C6 and C8. (A) HeLa cells were transiently transfected with pEGFP-C11 and pEGFP-C14. Coverslips were collected 48 hr post-transfection and stained with anti-PSP1 antibody. Both proteins showed colocalisation with PSP1 in at least some paraspeckles (arrows). (B) HeLa cells were transiently transfected with pEGFP-C6 and pEGFP-C8.

Coverslips were collected 48 hr post-transfection , after a 4-hr treatment with ActD, and stained with anti-PSP1 antibody. Both proteins showed colocalisation with PSP1 in perinucleolar caps (arrowheads).

Constructs C2, C4 and C6 are discussed in detail in the manuscript. To summarise, IF data from these three constructs showed that only the GFP-C6 protein localised in paraspeckles and perinucleolar caps. This indicates that neither the RNA nor the U2AF⁶⁵ binding domain of SF1 are sufficient for targeting the protein to paraspeckles and perinucleolar caps and suggests that this localisation depends mainly on sequences at the C-terminal end of the common region of the SF1 isoforms.

To test this possibility, the C-terminal regions corresponding to three SF1 isoforms (C7, C9, and C10) were transiently expressed in HeLa cells. All proteins contained residues encoded by common exon 10b. Proteins C7, C9 and C10 (corresponding to the C-terminal halves of SF1-GKS-Ala-A, SF1-Pro-A and SF1-GKS-Ala-B, respectively) were diffusely distributed in the nucleus, but were also detected in bright foci. The proteins distributed to perinucleolar caps after transcription inhibition. Mutant C8 was then examined, which contains only sequences encoded by exon 10b and which were included in all C-terminal mutants tested above. C8 was also present in paraspeckles and perinucleolar caps, but exhibited weak fluorescence in both structures. The latter result suggested that exon 10b-encoded residues are sufficient for localisation in both paraspeckles and perinucleolar caps. However, when these sequences were deleted from isoform SF1-GKS-Ala-B to yield mutant C21, the protein showed a distribution resembling that of full-length SF1, both in transcriptionally active and inactive cells. This indicates that sequences in exon 10b although sufficient, are not necessary for SF1 localisation in paraspeckles and perinucleolar caps.

IF data from transfections with mutant construct pEGFP-C16 showed that GFP-C16 (containing Ala-rich sequences encoded by exon 11b, 12 and 130 nt of exon 13) was not present in paraspeckles but, in transcriptionally inactive cells, it concentrated in perinucleolar caps. This finding suggests that, at least for the Ala-rich isoforms, a second perinucleolar cap-targeting signal exists. The contribution of exon 11b-encoded Ala-rich sequences in perinucleolar cap localisation was subsequently examined by analysis of mutant C19 that contains only exon 11b. GFP-C19 did not concentrate in the periphery of the nucleoli, implying that the second perinucleolar cap-targeting signal resides in sequences encoded by exon 12 and/or 130 nt of exon 13.

Analysis of more mutants (C11, C12, C13, C14, C15, C18) did not provide any supplementary information as to the targeting signal(s) involved in SF1 localisation to paraspeckles and perinucleolar caps.

Together, from these results we conclude that the RNA and U2AF⁶⁵ binding domains of SF1 are neither sufficient, nor necessary to target SF1 to paraspeckles or perinucleolar caps. Instead, it is evident that sequences encoded by exon 10b can target the protein to paraspeckles but are not necessary for this localisation. In addition, the results further indicate that, at least for the Ala-rich isoforms, a second perinucleolar cap-targeting signal exists, which resides in exon 12 and/or the first 130 nt of exon 13. In silico analysis of the sequences of exons 10b, 12 and 13 revealed no known motifs and no significant similarity to any other proteins.

Paraspeckles are probably not sites of splicing

Paraspeckles are probably not sites of splicing