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Mol Biol Cell
2011 Nov 01;2222:4312-23. doi: 10.1091/mbc.E11-07-0629.
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Functional analysis of the microtubule-interacting transcriptome.
Sharp JA, Plant JJ, Ohsumi TK, Borowsky M, Blower MD.
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RNA localization is an important mechanism for achieving precise control of posttranscriptional gene expression. Previously, we demonstrated that a subset of cellular mRNAs copurify with mitotic microtubules in egg extracts of Xenopus laevis. Due to limited genomic sequence information available for X. laevis, we used RNA-seq to comprehensively identify the microtubule-interacting transcriptome of the related frog Xenopus tropicalis. We identified ~450 mRNAs that showed significant enrichment on microtubules (MT-RNAs). In addition, we demonstrated that the MT-RNAs incenp, xrhamm, and tpx2 associate with spindle microtubules in vivo. MT-RNAs are enriched with transcripts associated with cell division, spindle formation, and chromosome function, demonstrating an overrepresentation of genes involved in mitotic regulation. To test whether uncharacterized MT-RNAs have a functional role in mitosis, we performed RNA interference and discovered that several MT-RNAs are required for normal spindle pole organization and γ-tubulin distribution. Together, these data demonstrate that microtubule association is one mechanism for compartmentalizing functionally related mRNAs within the nucleocytoplasmic space of mitotic cells and suggest that MT-RNAs are likely to contribute to spindle-localized mitotic translation.
FIGURE 1:. Purification and sequencing of MT-RNA. (A) Purification scheme to isolate MT-RNA. Eggs were harvested from female X. tropicalis frogs. After preparation of a cytoplasmic extract, Taxol was added to induce microtubule polymerization. Microtubules and MT-RNA were purified by sedimentation through a glycerol cushion. (B) Coomassie gel analysis of proteins isolated using the scheme described in A. Total CSF extract was compared with proteins sedimented in the presence of Taxol or nocodazole. (C) Bioanalyzer gel analysis of RNA isolated using the scheme described in A. RNA isolated from CSF extract was compared with RNA sedimented in the presence of Taxol or nocodazole. Both the gel projection and the line traces are shown. (D) RNA sequencing of CSF extract and MT-RNA. Sequences were aligned to the X. tropicalis genome and then compared with the Ensembl or RefSeq gene annotation databases to group sequences mapping to defined gene models. The scatter plots show read numbers per locus, plotted as a function of read number in CSF extract (x-axis) and read number in the MT-RNA fraction (y-axis). Data points that represent a value of 2 SDs above the mean log2(MT-RNA/CSF) ratio are plotted in red.
FIGURE 2:. RNA-seq data mapped to the incenp, tpx2, xkid, and stag1 loci. Peaks representing accumulated sequences (red) were plotted relative to existing gene models (blue). (A) The incenp gene on scaffold 306: 113565–123005. Peak scale represents 0–5547 reads. (B) The tpx2 gene on scaffold 2319: 3641–9330. Peak scale represents 0–4751 reads. (C) The xkid gene on scaffold 1489: 21516–28810. Peak scale represents 0–425 reads. (D) The stag1 gene in an unannotated region of scaffold 10: 2160151–2183813. Peak scale represents 0–109 reads.
FIGURE 3:. Bioinformatic and real-time PCR analysis of MT-RNA. (A) Venn diagram showing the degree of overlap for MT-RNAs identified from the Ensembl and RefSeq databases. A total of 454 unique MT-RNAs were identified by RNA-seq. (B) Quantitative real-time RT-PCR was performed on selected transcripts (cenpe, incenp, ckap2, tpx2, xrhamm, cep290, eg6, cspp1, stil, cenpj, talpid3, and smc1a) identified as MT-RNAs by RNA-seq to confirm microtubule association. MT-RNA was isolated from X. tropicalis eggs as described in Figure 1 and was compared with total RNA from CSF extract. Several transcripts that did not show selective enrichment on microtubules (ttll4, ranbp3, stag2) served as controls. Error bars represent the SE from the mean of three independently prepared extracts. (C) GO analysis was performed to determine whether any GO terms were dominant among the transcripts identified as MT-RNAs. Shown are the top 10–ranked GO terms using the annotation clustering function of the NIH DAVID database.
FIGURE 4:. MT-RNAs associate with spindle microtubules in vivo. X. laevis XL177 cells were fixed and processed for combined immunofluorescence for tubulin and FISH as described in the Materials and Methods. (A) The fluorescence hybridization signal for the MT-RNAs incenp, xrhamm, and tpx2. β-actin was used as a control. (B) The RNA signal merged with tubulin immunofluorescence and DAPI staining to visualize spindle microtubules and chromatin. Scale bar, 10 μm. (C) A selected area of the panels in B were enlarged four times. Scale bar, 2.5 μm.
FIGURE 5:. MT-RNAs are distinct from P-body domains. XL177 cells were labeled with antibodies to gw182 and fluorescent probes to detect the incenp, xrhamm, and tpx2 mRNAs. Scale bar, 10 μm.
FIGURE 6:. RNAi of MT-RNAs results in defective spindle pole organization. (A) RNAi was performed in HeLa cells to deplete transcripts for uncharacterized MT-RNAs. In control transfections, siRNAs for green fluorescent protein were used. Bar graph showing the percentage of HeLa cells displaying either abnormal spindle pole organization (red) or the formation of supernumerary spindle poles (blue). The error bars represent the SE from three independent experiments. (B) Images showing examples of spindles observed in RNAi experiments. HeLa cells were stained with an anti-tubulin antibody to label spindle microtubules, an anti–histone H3-S10P antibody to label chromatin of mitotic cells, and DAPI to label DNA for both interphase and mitotic cells. Scale bar, 10 μm.
FIGURE 7:. RNAi of MT-RNAs results in abnormal γ-tubulin distribution. (A) RNAi was performed in HeLa cells to deplete transcripts for the same MT-RNAs analyzed in Figure 4. Bar graph showing the percentage of HeLa cells with γ-tubulin dispersed throughout the cytoplasm. The error bars represent the SE from three independent experiments. (B) Images showing examples of γ-tubulin distribution observed in RNAi experiments. HeLa cells were stained with an anti-γ-tubulin antibody and DAPI to stain DNA. Scale bar, 10 μm.
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