COMMENTARY Where in the cell is the minor spliceosome? Joan A. Steitz*†, Gideon Dreyfuss‡, Adrian R. Krainer§, Angus I. Lamond¶, A. Gregory Materaʈ, and Richard A. Padgett** *Yale University and Howard Hughes Medical Institute, New Haven, CT 06536; ‡University of Pennsylvania School of Medicine and Howard Hughes Medical Institute, Philadelphia, PA 19104-6148; §Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; ¶University of Dundee, Dundee DD1 5EH, United Kingdom; ʈUniversity of North Carolina, Chapel Hill, NC 27599; and **Cleveland Clinic, Cleveland, OH 44195 he nucleus, a hallmark of spliceosome has subsequently been stud- of the LNA probes under the specific eukaryotes, segregates the ge- ied with respect to its protein composi- hybridization conditions used for cell nome and the cellular machin- tion and mechanism (8, 9), as well as its staining. Furthermore, control anti-U2 eries that transcribe and phylogenetic distribution and evolution- and anti-U5 probes did not show selec- Tprocess mRNAs from the translation ary origins (10). The minor spliceosome tive nucleoplasmic staining with nucleo- apparatus in the cytoplasm. Compart- excises Ϸ1 in 300 introns from human lar exclusion, as is well documented for mentalization prevents primary gene pre-mRNAs (11)—which encode pro- components of the major spliceosome transcripts (pre-mRNAs) from engaging teins with a wide range of functions (4, 16, 17). in translation before they are fully ma- (12)—consistent with the lower abun- Pessa et al. (2) performed in situ hy- tured. Thus, major mRNA processing dance (Ϸ1%) of its snRNPs relative to bridization on embryonic and adult Ј events—pre-mRNA splicing and 3 those of the major spliceosome. At least mouse tissue sections and on human polyadenylation—take place in the nu- seven proteins are unique to the minor cells, using full-length antisense cRNAs cleus. A recent paper by Ko¨nig et al. (1) spliceosome (13). GFP-tagged versions verified for specificity in Northern blots challenged this view, claiming that the of two—the U11/U12-associated pro- under identical conditions. They found splicing of hundreds of genes occurs in teins 35K and 31K (MADP-1)—were that both digoxigenin-labeled and radio- the cytoplasm, after export of unspliced reported to concentrate in the nucleus actively labeled probes for U11, U12, pre-mRNAs from the nucleus. This pa- of plant and HeLa cells, respectively U4atac, and U6atac snRNAs all strongly per sparked intense controversy. Be- (14, 15). label the nucleoplasm. Importantly, un- cause the central issue of whether Ko¨nig et al. (1) called this previous der the same conditions, probes specific mRNA formation and mRNA transla- work into question by localizing two mi- for the U1, U2, and U6 snRNAs of the tion occur in separate compartments is nor spliceosomal snRNAs in the cyto- major spliceosome exhibit strong nuclear so fundamental to eukaryotic cell biol- plasm of vertebrate cells and suggesting staining with nucleolar exclusion, as ogy, we examine here the experimental bases for this claim and contrast them expected. Comparable results were ob- with the findings of Pessa et al. (2), in tained by fluorescence in situ hybridiza- this issue of PNAS, which provide con- The minor spliceosome tion on HeLa cells, showing nearly complete overlap of the labeling for U2 tradictory evidence. excises Ϸ1 in 300 introns The vast majority of introns are and U12, and for U11 and U4, snRNAs. spliced by the major spliceosome: a from human pre-mRNAs. In their second approach, Ko¨nig et al. complex RNA–protein machine contain- (1) used cellular fractionation, followed ing the U1, U2, U4/U6, and U5 small by RT-PCR analysis, to reveal the pres- nuclear ribonucleoproteins (snRNPs). ence of unspliced minor introns in that the excision of minor introns takes Splicing can occur cotranscriptionally the cytoplasm. The extent of cross- place outside the nucleus, with func- and is generally thought to be com- contamination of the respective nuclear pleted in the nucleus before mRNA ex- tional and evolutionary implications. In and cytoplasmic fractions was only par- port. Although the biogenesis of four of response, Pessa et al. (2) present new in tially addressed. Data for three genes the major spliceosomal snRNPs (U1, situ hybridization and cell fractionation with minor introns clearly showed U2, U4, and U5) involves a cytoplasmic studies on the full set of minor snRNPs, spliced mRNA in the nuclear fraction. phase, the mature snRNPs and associ- extending prior evidence for the nuclear Although the authors attributed this to ated protein splicing factors ultimately location of the minor spliceosomal ‘‘the likely presence of material derived concentrate in the nucleus. snRNA and protein components. Al- from outer nuclear structures in the nu- A minor class of introns is removed though the two papers examine different clear fractions,’’ these results actually by a closely related machinery termed sets of cells and tissues, it is highly un- support the idea that minor-intron splic- the ‘‘minor spliceosome.’’ The U11 and likely that both conclusions can be ing occurs in the nucleus, although per- U12 snRNPs were described in 1988 as correct. haps not exclusively there. Indeed, when low-abundance particles similar to the U6atac was inactivated with an anti- Contrasting Data well characterized snRNPs of the major sense oligonucleotide, the minor-intron- spliceosome (3). In 1993, these particles Ko¨nig et al. (1) concluded that splicing spliced mRNAs in the nuclear fraction were localized, like their U1 and U2 of the minor class of introns is disappeared; it seems unlikely that all counterparts, by in situ hybridization to cytoplasmic on the basis of two main the spliced mRNA in the nuclear frac- the nucleoplasm of mammalian cells (4). experimental approaches. First, they In 1996, they were assigned functions in conducted in situ hybridization employ- the splicing of minor-class introns (5, 6), ing antisense locked nucleic acid (LNA) Author contributions: J.A.S., G.D., A.R.K., A.I.L., A.G.M., and and the U4atac and U6atac snRNAs of probes for U12 and U6atac snRNAs on R.A.P. wrote the paper. the minor spliceosome were identified embryonic and adult zebrafish tissues The authors declare no conflict of interest. (7). Thus, although four snRNPs are and on mouse 3T3 cells. In all cases, the See companion article on page 8655. unique to the minor spliceosome, the two probes showed predominantly cyto- †To whom correspondence should be addressed. E-mail: same U5 snRNP is shared by the minor plasmic signals. No Northern analyses [email protected]. and major spliceosomes. The minor were included to confirm the specificity © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804024105 PNAS ͉ June 24, 2008 ͉ vol. 105 ͉ no. 25 ͉ 8485–8486 Downloaded by guest on October 2, 2021 tion in untreated cells respresented cyto- complex (19). Subsequently, assembled version of the U4 snRNA can substitute plasmic contamination. snRNPs are reimported into the nu- in vivo for U4atac in the minor spliceo- Pessa et al. (2) also conducted cleus. The minor snRNAs contain all of some (24), which would not be possible nuclear/cytoplasmic fractionation of the signals required for nuclear import, if the two splicing events occurred exclu- HeLa cells and quantitated the relative including the m3G cap and an Sm site sively in separate cellular compartments. abundance of snRNA and protein com- bound by an apparently identical set of Similarly, mutation of a minor 5Ј splice ponents of the minor spliceosome. Sm proteins. Like the major snRNPs, site in a construct expressed in vivo effi- Although U6 and U6atac are approxi- the Sm core of the U11 snRNP is as- ciently activates an internal cryptic mately equally nuclear and cytoplasmic, sembled by the SMN complex (20), and major-class intron by weakening interac- presumably as a result of leakage of the minor U4atac and U6atac snRNPs tions with U11 snRNA (25); if minor- RNA polymerase III transcripts during possess protein compositions identical to intron splicing were cytoplasmic, why fractionation (18), the vast majorities of the major U4 and U6 snRNPs (21). wouldn’t this intron always be excised by the U11, U12, and U4atac snRNAs are Thus, for the conclusions of Ko¨nig et al. the major spliceosome before the minor nuclear and mirror the distributions of (1) to be correct, an as yet uncharacter- spliceosome encountered it? Moreover, U1, U2, and U4, respectively. Moreover, ized mechanism would be required to cytoplasmic mRNAs containing un- three additional proteins that are unique selectively slow or prevent the reimport spliced minor introns would be suscepti- to the minor spliceosome—U11-59K, of newly assembled minor snRNPs. ble to nonsense-mediated mRNA decay -35K, and -25K—are likewise predomi- Contrary to the conclusions of Ko¨nig (26), unless some selective mechanism nantly in the nuclear fraction, as re- et al. (1), much functional evidence existed to prevent their translation. vealed by Western blotting with poly- indicates that splicing of minor-class clonal antibody probes (13). Additional introns—like that of major-class introns— In conclusion, although science occa- RNA and protein controls exhibit their occurs predominantly, if not exclusively, sionally leaps forward when established established nuclear or cytoplasmic in the nucleus (8, 9). Minor-intron splic- principles are questioned, in this case we locations. ing can be reproduced in vitro in HeLa believe that the paradigm of nuclear nuclear extracts under conditions nearly pre-mRNA splicing has survived the Previous Findings identical to those used for major-intron recent challenge. The experiments of The results of Pessa et al. (2) strongly splicing (5) but does not occur in cyto- Pessa et al. (2) provide strong evidence support the large body of prior evidence plasmic extracts except upon addition of that casts doubt on the claim that the arguing for the nuclear location of splic- one or more nuclear components, as is minor spliceosome is localized in a dif- ing by both the major and minor splice- also the case for major-intron splicing ferent cellular compartment from the osomes.