A Whole-cell extracts (WCE) of HeLa cells transfected with non-targeting (CTRL) or UBL5 siRNAs for 48 h were immunoblotted with anti-UBL5 antibody.
B HeLa cells were counted at indicated times after transfection with control, UBL5, or SGO1 siRNAs [mean ± SD (error bars); N = 3].
C Sub-G1 DNA content of HeLa cells transfected with indicated siRNAs for 72 h was measured by flow cytometry analysis of propidium iodide-labeled cells [mean ± SD (error bars); N = 3].
D Mitotic indices of HeLa cells transfected with indicated siRNAs for 48 h were quantified by flow cytometry analysis of the proportion of phospho-H3(Ser10)-positive cells [mean ± SD (error bars); N = 3].
E Mitotic duration from nuclear envelope breakdown to chromatin decondensation in single live HeLa/H2B-mCherry cells transfected with control (n = 20) or UBL5 siRNA (n = 80) was monitored by time-lapse microscopy and classified as normal (45–60 min), delayed (longer than 60 min), or arrested (no decondensation).
F Time-lapse analysis of mitotic chromosome dynamics in live HeLa/H2B-mCherry cells transfected with control or UBL5 siRNA. Selected time frames of representative mitotic progression patterns and their frequencies are shown.
G Representative images of metaphase spreads from cells transfected with non-targeting (CTRL) or UBL5 siRNA. See also Supplementary Fig .
H Quantification of precocious sister chromatid separation in HeLa cells after knockdown of UBL5 [mean ± SD (error bars); N = 3]. At least 100 metaphase spreads were counted.
I HeLa cells inducibly expressing siRNA-resistant (si^R) Strep-HA-UBL5 were transfected with control or UBL5 siRNA in the absence or presence of doxycycline (DOX). Two days later, cells were treated with nocodazole for 3 h and harvested for chromosome analysis.

A Whole-cell extracts (WCE) of stable HeLa/Strep-HA-UBL5 cells induced or not with doxycycline (DOX) for 48 h were analyzed by immunoblotting with antibodies to HA and MCM6 (loading control).
B HeLa/Strep-HA-UBL5 cells treated as in (A) were fixed and immunostained with HA antibody. Scale bar, 10 μm.
C Mass spectrometry (MS)-based analysis of UBL5-interacting proteins. HeLa cells expressing Strep-HA-UBL5 or not were cultured in heavy (H) or light (L) SILAC medium, respectively. Strep-HA-UBL5 and associated proteins enriched on Strep-Tactin Sepharose were analyzed by MS. Plot shows Z-scores (from SILAC H/L ratios) and total intensity of identified proteins. Selected prospective UBL5 interactors associated with the spliceosome are highlighted. See Supplementary Table for full results.
D Selected proteins with high SILAC (H/L) ratios identified in the experiment in (C).
E Functional interactions of all proteins with a Z-score above 2.5 in the UBL5 pull-down experiment were obtained from the STRING database and visualized as a network. Node size corresponds to the Z-score. Known spliceosome components are highlighted in red.
F Gene ontology (GO) enrichment analysis of putative UBL5-interacting proteins. Significance of the enrichment is indicated for each term.
G Extracts of stable HeLa/Strep-HA-UBL5 cells induced or not with doxycycline (DOX) for 48 h were subjected to Strep-Tactin pull-down followed by immunoblotting with indicated antibodies.

A Proportion of reads spanning exon–intron junctions determined by RNA-Seq analysis of HeLa cells treated with control (CTRL), UBL5, or SART1 siRNAs for 48 h [mean ± SEM (error bars); N = 2]. Supplementary Table shows full RNA-Seq results.
B Density of RNA-Seq reads (pink) mapping to exons (yellow) and introns (blue) in the FASN gene, representative of genes displaying intron retention.
C Frequency of intron retention (IR) among all classified alternative splicing events, obtained via spliceR from RefSeq, Gencode, and UBL5 transcriptomes. Asterisk (*) denotes statistically significant changes (P < 2.2 × 10⁻¹⁶, Fisher’s exact test).
D Significantly differentially expressed transcripts were extracted and divided into the four indicated, overlapping subsets. The distribution of IF values from each condition was plotted. Asterisk (*) denotes changes that are statistically significant between control and all other samples (P < 1 × 10⁻⁴, pairwise Mann–Whitney U-test).
E Significantly differentially expressed transcripts were extracted, and the frequency of NMD sensitivity was compared between transcripts with and without IR. Asterisk (*) denotes statistically significant changes (P < 2.2 × 10⁻¹⁶, Fisher’s exact test).
F Whole-cell extracts (WCE) of HeLa cells transfected with non-targeting (CTRL) or UBL5 siRNAs for 48 h were immunoblotted with indicated antibodies.

A Precocious sister chromatid separation in HeLa cells transfected with indicated siRNAs [mean ± SD (error bars); N = 3]. At least 100 metaphase spreads were counted. Knockdown efficiency of siRNAs was analyzed by immunoblotting (Supplementary Fig ).
B Levels of cohesion factors in HeLa cells transfected with non-targeting (−) or UBL5 siRNAs were determined by immunoblotting.
C HeLa cells transfected with indicated siRNAs were processed for immunoblotting.
D Proportion of RNA-Seq reads from Sororin transcripts mapping to introns in cells transfected with indicated siRNAs [mean ± SEM (error bars); N = 2].
E Density of RNA-Seq reads (pink) mapping to exons (yellow) and introns (blue) in the Sororin (CDCA5) gene, showing retention of intron 1 (marked by red lines).
F Precocious sister chromatid separation in HeLa cells transfected with indicated siRNAs [mean ± SD (error bars); N = 3]. At least 100 metaphase spreads were counted. Knockdown efficiency of siRNAs was analyzed by immunoblotting (Supplementary Fig ).
G Sister chromatid cohesion status in metaphase spreads from cells transfected with indicated siRNAs and HA-Sororin cDNA [mean ± SD (error bars); N = 3]. Rescue efficiencies were normalized to the effect of expressing HA-Sororin in siSororin-treated cells. At least 100 metaphase spreads were counted. See also Supplementary Fig .