Grenfell AW et al. (2016), Mitotic noncoding RNA processing promotes kinet...

XB-ART-52295

J Cell Biol 2016 Jul 18;2142:133-41. doi: 10.1083/jcb.201604029.

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Mitotic noncoding RNA processing promotes kinetochore and spindle assembly in Xenopus.

Grenfell AW , Heald R , Strzelecka M .

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Transcription at the centromere of chromosomes plays an important role in kinetochore assembly in many eukaryotes, and noncoding RNAs contribute to activation of the mitotic kinase Aurora B. However, little is known about how mitotic RNA processing contributes to spindle assembly. We found that inhibition of transcription initiation or RNA splicing, but not translation, leads to spindle defects in Xenopus egg extracts. Spliceosome inhibition resulted in the accumulation of high molecular weight centromeric transcripts, concomitant with decreased recruitment of the centromere and kinetochore proteins CENP-A, CENP-C, and NDC80 to mitotic chromosomes. In addition, blocking transcript synthesis or processing during mitosis caused accumulation of MCAK, a microtubule depolymerase, on the spindle, indicating misregulation of Aurora B. These findings suggest that co-transcriptional recruitment of the RNA processing machinery to nascent mitotic transcripts is an important step in kinetochore and spindle assembly and challenge the idea that RNA processing is globally repressed during mitosis.

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Species referenced: Xenopus laevis
Genes referenced: frrs1 kif2c lrrfip1 mbp ndc80 tpx2 tubb2b

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	Figure 1. ncRNA biogenesis is important for mitotic spindle assembly. (A) Example images and quantification of spindle microtubule (MT) density and solidity after RNase H–based knockdown of the U2 snRNA (U2KD, n = 3,018) or treatment with a scrambled oligo (n = 1,454) in X. laevis egg extracts. Median microtubule density decreased 18.0% in U2 knockdown extract. (B) Example images and quantification of spindle microtubule density and solidity for X. tropicalis spindles formed after splicing factor immunodepletion with an antibody against the trimethylguanosine cap of snRNAs (α-TMG, n = 581) compared with mock depletion (IgG, n = 325). Median microtubule density decreased 24.5% in splicing factor-depleted extract. (C) Example images and quantification of spindle microtubule density and solidity in X. laevis extracts treated with splicing inhibitor (ISGN, n = 518), transcription inhibitor (TRIP, n = 417), or solvent control (DMSO, n = 483). Inhibitors were added immediately before spindle assembly. Median microtubule density decreased 14.9% in ISGN-treated extract and 24.1% in TRIP-treated extract. Bars, 10 µm. Box plot horizontal lines correspond to median values. Bottom and top of the boxes are first and third quartiles, respectively; whiskers show highest and lowest values within 1.5 times the interquartile range and outliers are plotted as single points. , P < 10−5; , P < 10−10; **, P < 10−15 (Kolmogorov–Smirnov test). AU, arbitrary units.
	Figure 2. The RNA processing machinery contributes to centromeric ncRNA biogenesis during mitosis. (A) Schematic of the centromere RNA biogenesis assay in X. laevis egg extract. Nuclei containing replicated sperm chromosomes were added to RNA-depleted extract, and nascent centromeric ncRNAs were assessed by RT-PCR. (B) Centromeric RNAs appeared in a laddered pattern after splicing factor depletion with an antibody specific to the trimethylguanosine cap of core spliceosomal RNAs (ID), whereas mock depletion (IgG) resulted in one or a few centromeric RNA products. The fcr1 repeat is 174 bp. Primers used to amplify this sequence were 106 bp apart. (C) Centromeric RNAs appeared in a laddered pattern after splicing inhibition with ISGN and were not detected after transcription inhibition with triptolide. (D) RT-PCR analysis of centromeric fcr1 ncRNAs in immunoprecipitates using nonspecific IgG antibodies (IgG Beads) or antibodies specific to the trimethylguanosine cap of core spliceosomal RNAs (TMG Beads), compared with extract supernatants (Sup.). (E) RT-PCR analysis of centromeric fcr1 ncRNAs in CENP-C immunoprecipitates (Beads) or extract supernatants (Sup.). An amplification product from a potentially processed transcript appears at ∼150 bp. Note that PCR-amplified centromeric RNAs varied in size, likely because predicted splice sites present in the degenerate fcr1 RNA sequence could lead to a variety of splice products because of heterogeneity of centromeric sequences.
	Figure 3. Perturbation of RNA biogenesis leads to centromere and kinetochore defects. (A) Example images of CENP-A staining and quantification of median fluorescence intensity within antibody marked foci after inhibition of splicing (ISGN, n = 9,544) or transcription (TRIP, n = 9,613) compared with DMSO controls (n = 5,203). Median CENP-A staining density decreased 25.4% in ISGN-treated extract and 37.8% in TRIP-treated extract. (B) Example images of CENP-C staining and quantification of median fluorescence intensity within antibody marked foci after inhibition of splicing (ISGN, n = 1,214) or transcription (TRIP, n = 1,160) compared with DMSO controls (n = 2,411). Median CENP-C staining density decreased 22.4% in ISGN-treated extract and 59.6% in TRIP-treated extract. (C) Example images of NDC80 staining and quantification of median fluorescence intensity within antibody marked foci after inhibition of spicing (ISGN, n = 1,499) or transcription (TRIP, n = 1,623) compared with DMSO controls (n = 1,515). Median NDC80 staining density decreased 10.0% in ISGN-treated extract and 19.9% in TRIP-treated extract. Bars, 10 µm. **, P < 10−10 (Kolmogorov–Smirnov test). In the merged image, microtubules are red, centromere proteins are green, and DNA is cyan. AU, arbitrary units.
	Figure 4. ncRNA-dependent MCAK regulation contributes to spindle integrity. (A) Example images and quantification of MCAK staining density after inhibition of splicing (ISGN, n = 100) or transcription (TRIP, n = 127) compared with the control (DMSO, n = 89). In the merged image, microtubules are red, MCAK is green, and DNA is blue. Median MCAK staining density increased 41.9% in ISGN-treated extract and 39.7% in TRIP-treated extract. (B) Quantification of MCAK staining density after treatment with buffer control (n = 466), αNDC80 antibodies (n = 477), α-amanitin (n = 350), or αNDC80 antibodies + α-amanitin (n = 262). Median MCAK staining density increased 9.8% in αNDC80-treated extract, 39.6% in α-amanitin–treated extract, and 43.8% in αNDC80 + α-amanitin–treated extract. (C) Example images of spindles formed under each of the conditions in B. Bar, 10 µm. (D) Quantification of spindle solidity under each of the conditions in C. (E) Quantification of spindle microtubule (MT) density under each of the conditions in C. Median microtubule density decreased 22.5% in αNDC80-treated extract, 33.4% in α-amanitin–treated extract, and 38.2% in αNDC80 + α-amanitin–treated extract. Bars, 10 µm. , P < 10−10; *, P < 10−15 (Kolmogorov–Smirnov test). AU, arbitrary units.
	Figure 5. Addition of TPX2 partially rescues spindle defects caused by perturbing ncRNA biogenesis. (A) Example images of TPX2 staining in control and inhibitor-treated reactions. In the merged image, microtubules are red, TPX2 is green, and DNA is cyan. Bar, 10 µm. (B) Plot showing the ratio of TPX2/tubulin intensity in spindles in control (n = 242) and inhibitor-treated reactions (ISGN n = 234, TRIP n = 120). The TPX2/tubulin ratio decreased 13.9% in ISGN-treated extract and 45.9% in TRIP-treated extract. (C) Example images of spindles assembled in extract after treatment with DMSO + 200 nM MBP (n = 193), ISGN + 200 nM MBP (n = 235), TRIP + 200 nM MBP (n = 199), ISGN + 200 nM TPX2 (n = 265), or TRIP + 200 nM TPX2 (n = 126). Bar, 10 µm. (D) Quantification of spindle microtubule density and spindle solidity under each of the conditions in C. Median microtubule density decreased 50.6% in MBP + ISGN–treated extract, 51.0% in MBP + TRIP–treated extract, 24.0% in TPX2 + ISGN–treated extract, and increased 26.9% in TPX2 + TRIP–treated extract. +, P < 0.05; , P < 10−5; , P < 10−10; **, P < 10−15 (Kolmogorov–Smirnov test).

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	Figure 1. ncRNA biogenesis is important for mitotic spindle assembly. (A) Example images and quantification of spindle microtubule (MT) density and solidity after RNase H–based knockdown of the U2 snRNA (U2KD, n = 3,018) or treatment with a scrambled oligo (n = 1,454) in X. laevis egg extracts. Median microtubule density decreased 18.0% in U2 knockdown extract. (B) Example images and quantification of spindle microtubule density and solidity for X. tropicalis spindles formed after splicing factor immunodepletion with an antibody against the trimethylguanosine cap of snRNAs (α-TMG, n = 581) compared with mock depletion (IgG, n = 325). Median microtubule density decreased 24.5% in splicing factor-depleted extract. (C) Example images and quantification of spindle microtubule density and solidity in X. laevis extracts treated with splicing inhibitor (ISGN, n = 518), transcription inhibitor (TRIP, n = 417), or solvent control (DMSO, n = 483). Inhibitors were added immediately before spindle assembly. Median microtubule density decreased 14.9% in ISGN-treated extract and 24.1% in TRIP-treated extract. Bars, 10 µm. Box plot horizontal lines correspond to median values. Bottom and top of the boxes are first and third quartiles, respectively; whiskers show highest and lowest values within 1.5 times the interquartile range and outliers are plotted as single points. , P < 10−5; , P < 10−10; **, P < 10−15 (Kolmogorov–Smirnov test). AU, arbitrary units.
	Figure 2. The RNA processing machinery contributes to centromeric ncRNA biogenesis during mitosis. (A) Schematic of the centromere RNA biogenesis assay in X. laevis egg extract. Nuclei containing replicated sperm chromosomes were added to RNA-depleted extract, and nascent centromeric ncRNAs were assessed by RT-PCR. (B) Centromeric RNAs appeared in a laddered pattern after splicing factor depletion with an antibody specific to the trimethylguanosine cap of core spliceosomal RNAs (ID), whereas mock depletion (IgG) resulted in one or a few centromeric RNA products. The fcr1 repeat is 174 bp. Primers used to amplify this sequence were 106 bp apart. (C) Centromeric RNAs appeared in a laddered pattern after splicing inhibition with ISGN and were not detected after transcription inhibition with triptolide. (D) RT-PCR analysis of centromeric fcr1 ncRNAs in immunoprecipitates using nonspecific IgG antibodies (IgG Beads) or antibodies specific to the trimethylguanosine cap of core spliceosomal RNAs (TMG Beads), compared with extract supernatants (Sup.). (E) RT-PCR analysis of centromeric fcr1 ncRNAs in CENP-C immunoprecipitates (Beads) or extract supernatants (Sup.). An amplification product from a potentially processed transcript appears at ∼150 bp. Note that PCR-amplified centromeric RNAs varied in size, likely because predicted splice sites present in the degenerate fcr1 RNA sequence could lead to a variety of splice products because of heterogeneity of centromeric sequences.
	Figure 3. Perturbation of RNA biogenesis leads to centromere and kinetochore defects. (A) Example images of CENP-A staining and quantification of median fluorescence intensity within antibody marked foci after inhibition of splicing (ISGN, n = 9,544) or transcription (TRIP, n = 9,613) compared with DMSO controls (n = 5,203). Median CENP-A staining density decreased 25.4% in ISGN-treated extract and 37.8% in TRIP-treated extract. (B) Example images of CENP-C staining and quantification of median fluorescence intensity within antibody marked foci after inhibition of splicing (ISGN, n = 1,214) or transcription (TRIP, n = 1,160) compared with DMSO controls (n = 2,411). Median CENP-C staining density decreased 22.4% in ISGN-treated extract and 59.6% in TRIP-treated extract. (C) Example images of NDC80 staining and quantification of median fluorescence intensity within antibody marked foci after inhibition of spicing (ISGN, n = 1,499) or transcription (TRIP, n = 1,623) compared with DMSO controls (n = 1,515). Median NDC80 staining density decreased 10.0% in ISGN-treated extract and 19.9% in TRIP-treated extract. Bars, 10 µm. **, P < 10−10 (Kolmogorov–Smirnov test). In the merged image, microtubules are red, centromere proteins are green, and DNA is cyan. AU, arbitrary units.
	Figure 4. ncRNA-dependent MCAK regulation contributes to spindle integrity. (A) Example images and quantification of MCAK staining density after inhibition of splicing (ISGN, n = 100) or transcription (TRIP, n = 127) compared with the control (DMSO, n = 89). In the merged image, microtubules are red, MCAK is green, and DNA is blue. Median MCAK staining density increased 41.9% in ISGN-treated extract and 39.7% in TRIP-treated extract. (B) Quantification of MCAK staining density after treatment with buffer control (n = 466), αNDC80 antibodies (n = 477), α-amanitin (n = 350), or αNDC80 antibodies + α-amanitin (n = 262). Median MCAK staining density increased 9.8% in αNDC80-treated extract, 39.6% in α-amanitin–treated extract, and 43.8% in αNDC80 + α-amanitin–treated extract. (C) Example images of spindles formed under each of the conditions in B. Bar, 10 µm. (D) Quantification of spindle solidity under each of the conditions in C. (E) Quantification of spindle microtubule (MT) density under each of the conditions in C. Median microtubule density decreased 22.5% in αNDC80-treated extract, 33.4% in α-amanitin–treated extract, and 38.2% in αNDC80 + α-amanitin–treated extract. Bars, 10 µm. , P < 10−10; *, P < 10−15 (Kolmogorov–Smirnov test). AU, arbitrary units.
	Figure 5. Addition of TPX2 partially rescues spindle defects caused by perturbing ncRNA biogenesis. (A) Example images of TPX2 staining in control and inhibitor-treated reactions. In the merged image, microtubules are red, TPX2 is green, and DNA is cyan. Bar, 10 µm. (B) Plot showing the ratio of TPX2/tubulin intensity in spindles in control (n = 242) and inhibitor-treated reactions (ISGN n = 234, TRIP n = 120). The TPX2/tubulin ratio decreased 13.9% in ISGN-treated extract and 45.9% in TRIP-treated extract. (C) Example images of spindles assembled in extract after treatment with DMSO + 200 nM MBP (n = 193), ISGN + 200 nM MBP (n = 235), TRIP + 200 nM MBP (n = 199), ISGN + 200 nM TPX2 (n = 265), or TRIP + 200 nM TPX2 (n = 126). Bar, 10 µm. (D) Quantification of spindle microtubule density and spindle solidity under each of the conditions in C. Median microtubule density decreased 50.6% in MBP + ISGN–treated extract, 51.0% in MBP + TRIP–treated extract, 24.0% in TPX2 + ISGN–treated extract, and increased 26.9% in TPX2 + TRIP–treated extract. +, P < 0.05; , P < 10−5; , P < 10−10; **, P < 10−15 (Kolmogorov–Smirnov test).