Characterization and in vivo functional analysis of the Schizosaccharomyces pombe ICLN gene

Abstract

During the early steps of snRNP biogenesis, the survival motor neuron (SMN) complex acts together with the methylosome, an entity formed by the pICln protein, WD45, and the PRMT5 methyltransferase. To expand our understanding of the functional relationship between pICln and SMN in vivo, we performed a genetic analysis of an uncharacterized Schizosaccharomyces pombe pICln homolog. Although not essential, the S. pombe ICln (SpICln) protein is important for optimal yeast cell growth. The human ICLN gene complements the Δicln slow-growth phenotype, demonstrating that the identified SpICln sequence is the bona fide human homolog. Consistent with the role of human pICln inferred from in vitro experiments, we found that the SpICln protein is required for optimal production of the spliceosomal snRNPs and for efficient splicing in vivo. Genetic interaction approaches further demonstrate that modulation of ICln activity is unable to compensate for growth defects of SMN-deficient cells. Using a genome-wide approach and reverse transcription (RT)-PCR validation tests, we also show that splicing is differentially altered in Δicln cells. Our data are consistent with the notion that splice site selection and spliceosome kinetics are highly dependent on the concentration of core spliceosomal components.

FIG 1

The SpICln protein is a functional human ortholog and is not essential for viability. (A) Sequence alignment of pICln homologs, with gene identification (gi) numbers. The alignment was created using the PRALINE server (62). Identical amino acids among pICln proteins are shaded in black, similar residues are shaded in gray, and acidic domains are labeled. The secondary structures predicted by PSIPRED (position-specific iterated prediction of protein secondary structure) through the Ali2D server (University of Tübingen, Tübingen, Germany) are indicated below the sequences. The α-helix and the β₁- and β₇-strands (represented by arrows) correspond to the canonical pleckstrin homology domains (18, 33). Proposed highly (+) and strictly (*) conserved residues (18) are also shown. H. sapiens, Homo sapiens; M. musculus, Mus musculus; C. elegans, Caenorhabditis elegans; D. melanogaster, Drosophila melanogaster. (B) Tetrad analysis of diploid S. pombe cells with one deleted copy of ICLN (SPAC1610.01/SPAC1610.01::KanMX4). After sporulation, separated spores were grown on YES plates at 25°C for 5 days (the spores for each tetrad are arranged vertically). (C) Δicln cells have a growth defect and are temperature sensitive. Wild type (wt) and Δicln cells were grown in YES medium, and serial dilutions were spotted on YES plates, which were incubated at the indicated temperatures. (D) The growth defect of Δicln cells is complemented by the human ICln protein. Full-length cDNAs for the S. pombe and human ICLN genes were placed under the control of the nmt1+ promoter in a pREP41 plasmid. Transformants carrying the plasmids indicated on the right were grown in −Leu medium, and an equivalent number of cells were serially diluted, plated on −Leu plates, and incubated at 25°C for 5 days. (E) The indicated cells were transformed with the empty pREP41 vector, a plasmid encoding fission yeast ICLN, or a plasmid carrying the S. pombe SMN gene, and cultures of comparable density were serially diluted, spotted on EMM-Leu plates, and incubated at 25°C for 5 days. (F) Synthetic lethality between ICLN and SMN alleles. The Δicln::KanMX4 strain was crossed with the tdSMN::KanMX4 strain, and the resulting diploid was sporulated. Tetrads were dissected and incubated on YES medium at 25°C for 5 days. The segregation patterns (TT, tetratype; PD, parental ditype; NPD, nonparental ditype) of representative tetrads are shown. Nonviable spores are circled and are all Δicln tdSMN double mutant.

FIG 2

SpICln associates with Sm proteins in vitro and in vivo. (A) Glutathione-Sepharose beads and the indicated recombinant GST-Sm fusion proteins or GST alone was incubated with 1 μg of recombinant His₆-tagged SpICln protein. The beads were washed, and bound proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After S-Ponceau staining to view the GST fusions, the membrane was probed with an anti-His antibody to visualize the His-SpICln protein (bottom). (B) Coimmunoprecipitation of TAP-SpICln and GFP-SmD1 proteins. Extracts prepared from Δicln cells carrying the indicated plasmids were incubated with calmodulin binding protein resin. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-PAP and anti-GFP antibodies. Inputs (2%) are shown on the left. (C) Whole-cell extract prepared from Δicln cells carrying TAP-SpICln and GFP-SmD1 plasmids was separated on a 5 to 20% sucrose gradient. The direction of sedimentation (Sed) is top to bottom, as indicated by the arrow. After collection of the fractions, the odd-numbered fractions were separated on SDS-PAGE and immunoblotted to detect TAP-SpICln and GFP-SmD1 fusion proteins. (D) Fractions 3 to 5 from the sucrose gradient were pooled and incubated with calmodulin binding protein (CBP) resin or glutathione-Sepharose (GS) beads (as a control [Ctl]). The bound proteins were separated by SDS-PAGE and immunoblotted with anti-PAP and anti-GFP antibodies. Input represents 2% of the pooled fractions used in the binding assay.

FIG 3

The levels of snRNPs are decreased in fission yeast Δicln cells. (A) RNA was extracted from Δicln and wild-type cells grown at 25°C, separated on a 6% polyacrylamide–8 M urea gel, and subjected to Northern analysis. Hybridization was performed using probes specific for the indicated RNAs. (B) Quantitation of the Northern blot shown in panel A by scanning densitometry of the RNAs present in Δicln and wild-type cells. The error bars indicate standard deviations. (C) Analysis of snRNPs in Δicln (Δ) and wild-type cells by native gel electrophoresis. Extracts were prepared from cells grown at 25°C, and aliquots were separated on 4% native gels. The RNA was subjected to Northern blot analysis and hybridized with probes for the indicated RNAs. *, the U2/U5/U6 postsplicing complex; ×, the U4/U6 di-snRNP. (D) Splicing is inhibited in Δicln cells. Total RNA was isolated from Δicln and wild-type cells and used for primer extension with a ³²P-labeled primer specific to the U6 gene. Pre, species corresponding to the U6 precursor that contains an intron; mature, the spliced U6 RNA.

FIG 4

Differential splicing defects in Δicln cells revealed by microarray analyses. (A) Tiling array profiles of genes showing changes in intron signals in the Δicln cells compared to wild-type cells. The scale on the y axis represents the log₂ fold change between the treatment (Δicln) and control (wt) group signals. RT-PCR validation tests of the genes shown on the left were performed on total RNA isolated from the wild-type and Δicln cells grown at 25°C. Exon-specific primers were used to amplify the corresponding spliced and unspliced species. The PCR products were separated on 1.5% to 2% agarose gels and visualized by ethidium bromide staining. The gene systematic identifier and the intron numbers are indicated below the gel, and the schematics of spliced and intron-containing mRNAs are shown to the left of the gel. (B) The retained introns contain PPTs located further upstream of the branch point. The histogram depicts the frequencies of PPTs of 5 or more pyrimidines at the indicated positions upstream of the branch point adenosine in the group of 250 introns compared to the remaining 4,361 introns. The circled numbers represent positions having significant different frequencies (see text for more details). The Wilcoxon P value is indicated. (C) The retained introns contain a lower percentage of A/U. The histograms show a comparison of the base compositions (percent A/U) of the group of retained introns (group 250) and the remaining group of introns (group 4361) (t test; P = 2.2 × 10⁻¹⁶).