Will the real splicing sites please light up?

SUI HUANG AND DAVID L. SPECTOR

Will the real splicing

NUCLEARORGANIZATION

sites please light up?

RNA transcripts and splicing factors are associated with nuclear regions that can be visualized as speckles. Gene transcription first produces pre-mRNA transcripts, which are processed to mRNA in the nucleus and then transported to the cytoplasm, where they are translated into proteins. For most RNA polymerase II transcripts, the nuclear processing includes splicing of non-coding intron regions and subsequent ligation of exons. Splicing occurs in a m&component complex termed a spiiceosome. Much progress has been made towards a biochemical understanding of the basic mechanism of splicing and the functional components of the spliceosome (recently reviewed in [ 11). But where in the nucleus does the splicing take place? Despite some controversy, a number of recent cytological studies have advanced our understanding of the nuclear organization of splicing components and the relationship of their localization to the distribution of pre-mRJ?IA substrates. Small nuclear ribonucleoprotein particles (snRNps) were the Iirst components shown to be essential for pre-mRNA splicing (reviewed in [ 11). Each of the snRNP parti cles (Ul, U2, ~6) contains a single snRNA species with the exception of the U4/UG particle which contains two snRNAs. In addition, each snRNp particle contains a common set of core proteins, as well as some unique snRNAspecific proteins (reviewed in [ 21). The sub-localization of splicing components in the nucleus was first indicated by immunofluorescent localization studies using antibodies that recognize protein or RNA components of snRNPs. These studies revealed snRNPs to be concentrated in 2&50 nuclear speckles in addition to being dif fusely distributed in the nucleoplasm [ 3,4]. Subsequently, SC-35, a non-sr&NP spliceosome component essential for the early steps of pre-mRNA splicing (51, was found to co-localize with snRNPs, in a speckled pattern, with extensive connections between the speckles forming a three-dimensional network throughout the nucleus 161. The speckled distribution of SC-35 has been shown by immunoelectron microscopy to correspond to interchromatin granules and perichromatin fibrils [6]; electron microscopy had previously shown snRNp.s to be associated with both of these subnuclear components [7,8]. Interchromatin granules are thought to contain RNA species with a low turnover rate, whereas perichromatin fibrils, which are distributed throughout the nucleus, are thought to represent the sites of active transcription (reviewed in [9]). The presence of snRNP antigens 171, hnRNp antigens [7] and SC-35 [G] at these fibrils suggests that the splicing of pre-mRNA may occur at, or in very close proximity to, the sites of transcription. The close spatial association of splicing and transcription sites has also been suggested by other studies (reviewed in [ 10 ] >.

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The functional significance of the sub-localization of splicing components to speckled nuclear regions has recently been enhanced by the identification of a specific targeting signal. Li and Bingham [ll] showed that an arginine/serine(RS)-rich domain, composed of approximately 120 amino acids, from two different Drosophila pre-mRNA splicing regulators, suppressor-oj whiteapricot (su(w”)) and transformer (rraj, could tar get these proteins into the nucleus and localize them to speckled regions. Furthermore, the fusion of an ES domain to P-galactosidase directs this protein to the speckle region, suggesting that the ES domain is essential and sufficient to target proteins to these subnuclear regions Several other splicing factors, including tra2 El21 in DrosopM.a and SC-35 (X-D Fu and T Maniatis, personal communication), SF2/ASF [13,14], U2AF [l] and the Ul snRNP 70 kD polypeptide in mammalian cells [15] also contain an ES-rich domain. This domain may have a role in concentrating splicing factors in a sub-compartment of the nucleus (speckles) in order to favor interactions between splicing factors and pre-mRNA substrates, thus increasing the efficiency of spliceosome assembly, RNA cleavage and ligation. Because numerous previous studies had shown snRNP antigens, as well as SC-35, to be localized to a speckled nuclear pattern, it was quite surprising when two recent reports [16,17] suggested that the distribution of various snRNAs, including U2, U4/U6 and U5, was restricted to three or four ‘foci’ in the nucleus, while Ul snRNA was dilfusely distributed throughout the nucleoplasm in addition to being present in foci. These studies were performed by in situ hybridization using biotinconjugated 2’-O-methyl or alkyl antisense RNA oligomers as probes. This apparent discrepancy has recently been resolved. Using the same probes as in 1171 but extending the length of hybridization to 16 hours, we have re-examined the distribution of Ul and U2 snRNAs in various cell types by in situ hybridization [ 181. When HeIa cells were analysed in this manner, both Ul and U2 snRNAs were found to be concentrated in a speckled nuclear pattern in all cells but about 80% of the cells also showed labeling of foci (Fig. la). Depending upon the cell type examined, varying degrees of diffuse staining were also observed. The distribution of Ul and U2 snRNAs in speckles and foci coincided with the staining pattern obtained by anti-.snRNP antibodies. In contrast, SC-35, although present in speckles, was conspicuously absent from foci [18] and foci were not observed in a majority of cells with defined passage numbers (unpublished data) (Fig. lb).

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Biology

Fig. 1. Confocal laser scanning microscopy of snRNPs in the nucleus of (a) a HeLa cell (b) a Detroit 551 cell of defined passage Both have speckles but only the HeLa cell has foci (arrow). Interconnections between speckles and connections to the nuclear envelope can be observed.’

As foci are not present in all cells of a given population, and are rarely observed (l-5%> in cell types with deftned passage numbers, it is unlikely that they play an essential part in pre-mRNA splicing. By electron microscopy, we have identified the foci, which are intensely stained for snRNAs and snRNPs in HeIa cells, as coiled bodies (unpublished data), which are electron-dense nuclear structures enriched in snRNP antigens, snRNAs, the U3 specific protein librillarin, the recently identified coiled bodyspecific protein coilin [19], and possibly other unidentified components, The significance of the presence or absence of coiled bodies in cell nuclei is unclear. Zamore and Green 1201, using an anti-peptide antibody, showed that the non-snRNP splicing factor, U2AP, was both localized to foci and had a diffuse distribution. However, when an antibody against a bacterially expressed human U2AP protein is used in an immunofluorescence assay, U2AP is observed to be localized to speckles (J Galceran, J Patton and B Nadal-Ginard, personal communication). This localization fits with the fact that U2AF contains the type of P&rich domain that targets proteins to the speckled region. . Why are numerous splicing factors concentrated in speckled subnuclear regions? It has been speculated that pre-mRNAs may be spliced within or in close proximity to these regions, in which case pre-mRNAs might also be localized to speckles. Recently, three papers have provided evidence to support this hypothesis. Wang et al. [ 211 showed that when fluorescently-tagged P-globin pre-mRNA is microinjected into interphase nuVolume

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number. lamina-

clei it becomes localized in a speckled distribution coincident with the speckled pattern enriched in splicing factors. In contrast, microinjection of anti-sense RNA or of transcripts either lacking an intron or, with a deleted 3’ splice site, yields a diffuse distribution. Thus, introncontaining transcripts have the ability to associate with nuclear speckles enriched in pre-mRNA splicing factors. More recently, Carter et al. [22] have examined the localization of endogeneous poly(A) RNA in interphase nuclei by in situ hybridization. This study revealed a speckled distribution of poly(A) RNA that co-localized with the entire speckled pattern of snRNPs. This is puzzling because it has been shown that a portion of the speckled pattern (interchromatin granule clusters) contains little or no newly synthesized RNA (see [9]). It is possible that the portion of the poly(A) RNA associated with perichromatin fibrils is spliced and exported, whereas the other portion may remain in the nucleus and potentially have a structural role in interchromatin granule clusters. To analyse the localization of specific endogenous cellular transcripts and to elucidate the pathways that these transcripts take from their sites of synthesis to the nuclear envelope, we have localized nascent RNA transcripts of an inducible gene, c-fos, in the interphase nucleus by in situ hybridization [ 231. Confocal laser scanning and high voltage electron microscopy demonstrated that c-fos RNA transcripts extend as an elongated path from their sites of synthesis to the nuclear envelope. Double labeling of c-fos RNA transcripts and SC-35 revealed a close association between the c-j& RNA transcripts and the speckled regions. Taken together, these three studies [21-231 189

showing a close association of RNA substrates or products with nuclear regions enriched in splicing factors (speckles) provide the strongest support so far that these nuclear regions are involved in pre-mRNA splicing. On the basis of these recent findings, a tentative model of the nuclear organization of the pre-mRNA splicing apparatus is beginning to emerge. Actively transcribing genes are distributed throughout the nucleoplasm [9,24]. Upon activation of a gene, splicing factors from a nearby interchromatin granule cluster (storage and/or assembly sites) may move to the site of the active gene and associate directly with the nascent transcripts, which are visualized as perichromatin fibrils. The perichromatin fibrils may represent connections between larger speckles and may also be found in close proximity to the surface of the speckles. The association of splicing factors with nascent RNA transcripts supports the view that pre-n-&WA splicing occurs in close proximity to the sites of transcription. Therefore, the organization of the speckled pattern is a reflection of the transcriptional activity of the cell. Aclmowl&lgements We appreciate the helpful comments of Scott Hendersok; Luis Jiienez-Garcia, Adrian Kramer, Tom Maniatis, Raymond O’Keefe and Mona Spector. DLS is supported by grants from the Nati??pal Institutes of Health. ’

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Sui Huang and David L. Spector, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA

@ 1992 Current Biology