1. Volume 26, Issue 11, Pages 2929-2941.e5 (March 2019)
Functional Interaction between U1snRNP and Sam68 Insures Proper 3′ End Pre-mRNA Processing during Germ Cell Differentiation 
Chiara Naro, Livia Pellegrini, Ariane Jolly, Donatella Farini, Eleonora Cesari, Pamela Bielli, Pierre de la Grange, Claudio Sette 
Cell Reports 
Volume 26, Issue 11, Pages e5 (March 2019)
DOI: /j.celrep
Copyright © 2019 The Author(s) Terms and Conditions

2. Cell Reports 2019 26, 2929-2941.e5DOI: (10.1016/j.celrep.2019.02.058)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1
Sam68-Mediated Genome-wide Regulation of Male Germ Cells Transcriptome
(A) Pie charts showing percentages of differentially splicing-regulated exons identified in Sam68−/− versus Sam68+/+ sp.cytes (left) and sp.tids (right; fold change ≥ 2, p ≤ 0.05).
(B) Venn diagram showing the significant overlap between regulated splicing events in Sam68−/− versus Sam68+/+ sp.cytes and sp.tids (modified Fisher’s test).
(C) Pie chart showing percentages of up- and downregulated events among the commonly regulated events in Sam68−/− versus Sam68+/+ sp.cytes and sp.tids.
(D) Venn diagrams showing overlap between transcriptional- and splicing-regulated genes, identified by comparison of Sam68−/− versus Sam68+/+ sp.cytes (left panel) and sp.tids (right; for gene expression fold change ≥ 1.5 and p ≤ 0.05 were considered; p value indicated below Venn diagrams were calculated by modified Fisher’s test).
(E) Pie charts representing distribution of regulated splicing events among different splicing patterns in Sam68−/− versus Sam68+/+ sp.cytes (left) and sp.tids (right).
(F) Representative images of RT-PCR analyses for indicated alternative splicing (AS) events differentially regulated between Sam68−/− and Sam68+/+ male germ cells. Schematic representation for each event analyzed is depicted above the representative agarose gel. Red and green boxes indicate, respectively, up- and downregulated events in Sam68−/− versus Sam68+/+ male germ cells. Black arrows in the scheme indicate primers used for the PCR analysis. Relative ratio of indicated splice variants was evaluated by densitometric analysis, and results are shown below agarose gels (mean ± SD, n = 3, p values were calculated according to paired t test).
See also Figure S1.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

4. Figure 2
Sam68 Regulates Alternative Last Exon Selection in Differentiating Male Germ Cells
(A) Bar graph showing percentages of events annotated in FAST-DB (white columns) and of those differentially regulated between Sam68−/− versus Sam68+/+ male germ cells (black columns) within each AS pattern. p values above the graph indicate a significant enrichment for regulated events within a specific AS pattern in our dataset compared with the reference database (modified Fisher’s test). Red box highlights alternative terminal exon splicing pattern.
(B) Bar graph showing the enrichment score of the GO functional clusters enriched in the group of alternative last exon regulated genes between Sam68−/− and Sam68+/+ male germ cells (p ≤ 0.05, high stringency setting). Red bars indicate spermatogenesis-related clusters.
(C) Venn diagram showing overlap of regulated ALEs in the indicated comparisons; p value was calculated by modified Fisher’s test. ALEs regulated between Sam68+/+ spermatocytes and spermatids were previously identified (Naro et al., 2017).
(D) Bar graphs showing results of qPCR analyses for the expression of indicated ALE in Sam68−/− versus Sam68+/+ and in Sam68+/+ sp.cytes ad sp.tids, relative to the not regulated last exon (mean ± SD, n = 3; ∗p ≤ 0.05, ∗∗p ≤ 0.01, paired t test). Inset shows western blot analysis of Sam68 in Sam68+/+ sp.cytes and sp.tids.
See also Figure S2.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

5. Figure 3 Sam68 Prevents Usage of Premature Cryptic ALE
(A) Curve graph showing distribution of up- (red line, n = 122) and downregulated (green line, n = 413) alternative last exons along gene body (5′→3′).
(B) Bar graph representing percentages of “internal” (light gray) and “last” (black) ALE among up- and downregulated events. p value above the graph indicates a significant difference in the distribution of “internal” and “last” ALE between the two groups (Χ2 test).
(C) Visualization of the RNA-seq reads profile of regulated ALE within Ppp3cc and Spag6 genes in Sam68−/− versus Sam68+/+ male germ cells. Sequence reads (blue gray lines), regulated (red boxes) and non-regulated exons (gray boxes), and introns (horizontal lines) are shown. Dashed red boxes highlight sequence reads of regulated ALE.
(D) PCR analyses for indicated mRNA regions of Ppp3cc and Spag6 genes on G/I tailed RT samples. Genomic DNA (gDNA) and random hexamers primed RT (pdN6) samples were used as control of the PCR reaction. Gosr2 expression was evaluated as loading control.
(E) Bar graphs showing results of qPCR analyses for the relative expression of indicated regulated and canonical last exons on G/I tailed RT samples in Sam68+/+ and Sam68−/− male germ cells (mean ± SD, n = 3; ∗p ≤ 0.05, ∗∗p ≤ 0.01, paired t test).
(F) Chromatograms of sequencing reaction of 3′RACE-products for indicated Ppp3cc and Spag6 cryptic ALE. Red dashed boxes highlight alternative polyadenylation signals.
(G) Western blot analysis for PPP3CC and SPAG6 expression in Sam68−/− and Sam68+/+ male germ cells. Sam68 expression was probed as control of genotyping and ACTIN was evaluated as loading control.
See also Figure S3.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

6. Figure 4 Sam68 Binds Regulated Alternative Last Exon
(A) Logos representing consensus sequences for Sam68 binding motif (left logo) and the most common mammalian polyadenylation signal (PAS; right logo).
(B) Representative western blot analysis for CLIP experiments performed from 25 dpp mouse testis using Sam68 specific and control immunoglobulin G (IgG) antibody.
(C) Bar graphs showing results of qPCR analyses for indicated pre-mRNA regions of Agbl3, Ppp3cc, and Spag6 genes co-precipitated by Sam68-specific and control IgG antibody in CLIP experiments performed from 25 dpp mouse testis (mean ± SD, n = 3; ∗∗p ≤ 0.01; one-way ANOVA, followed by Sidak multiple comparisons test, only significant comparisons are indicated). Schematic representation of each gene, indicating position of non-regulated internal exons, distal last exons (gray boxes), and regulated internal last exons (red boxes) and of primers used for the analysis (black arrows) are depicted above each graph.
(D) Bar graph showing the percentage of sequences among upregulated (n = 152), downregulated (n = 139), and reference ALE (n = 444) having Sam68 motifs in their sequences. p values above the graph indicate a significant difference in the proportion of sequences having Sam68 motifs within the groups (Χ2 test).
(E) Bar graph showing proportion of sequences having Sam68 motifs in their sequence according their relative position (Intron N-1, upstream intron; Exon N, exonic ALE; AE N, intronic ALE; Intron N, downstream intron) among upregulated (n = 152), downregulated (n = 139), and reference ALE (n = 444). p values above the graph indicate a significant difference in the distribution of Sam68 motifs within the groups (Χ2 test).
See also Figure S4.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

7. Figure 5 U1snRNP Binding Sites Are Enriched within Sam68-Regulated ALE
(A) Schematic representation of the U1snRNP. U1 snRNA and associated RNA-binding proteins are indicated.
(B) Logo representing consensus sequence for U1 binding tested for motif analysis enrichment.
(C) Boxplot graph representing distribution of the distances between U1 and Sam68 binding motifs among upregulated (n = 152), downregulated (n = 139), and reference ALE (n = 444). Whiskers indicate 1.5 interquartile range (p values refer to the differences between mean values estimated with a t test).
See also Figure S5.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

8. Figure 6
Sam68 Interacts and Cooperates with U1snRNP in Alternative Last Exon Selection
(A–C) Representative western blot analysis for indicated proteins of immunoprecipitation assay in 25 dpp testes extracts using control IgG or anti-Smith antigen (A), anti-Sam68 (B), and anti-U1-C (C) antibodies.
(D) Bar graph showing results of qPCR analyses for indicated snRNAs co-precipitated by Sam68-specific and control IgG antibody in RNA immunoprecipitation (RIP) experiments performed from 25 dpp mouse testis (mean ± SD, n = 3; ∗p ≤ 0.05; one-way ANOVA, followed by Sidak multiple comparisons test, only significant comparisons are indicated).
(E) Bar graph showing results of qPCR analyses for indicated mRNAs co-precipitated by anti-Smith antigen and control IgG antibody in RIP experiments performed from 25 dpp mouse testis, normalized to the amount of precipitated U1 snRNA (mean ± SD, n = 3; ∗p ≤ 0.05; t test).
(F) Scheme of wild-type (WT) and mutant Ppp3cc minigenes, carrying mutations for either Sam68 binding motifs (mut Sam68) or partial deletion of U1 binding motif (ΔU1). Polyadenylation signal and Sam68 and U1 binding motifs are indicated, and mutated bases within Sam68 motifs are in red.
(G) Results of qPCR analyses for the relative expression of transcripts including exon 14 or exon 16 in splicing assay in HEK293T cells overexpressing Sam68 and transfected with WT or mut Sam68 Ppp3cc minigene. Data were normalized to the relative ratio obtained for either WT or mutant minigene in cells transfected with empty vector, set to 1 (mean ± SEM, n = 4; ∗p ≤ 0.05, ∗∗p ≤ 0.01, paired t test).
(H) Results of qPCR analyses for the relative expression of transcripts including exon 14 or exon 16 in splicing assays in HEK293T cells silenced for Sam68 and transfected with WT or ΔU1 Ppp3cc minigene. Data were normalized to the relative ratio obtained for either WT or mutant minigene in cells transfected with control small interfering RNA (siRNA; set to 1 and indicated by dashed line in the graph; mean ± SEM, n = 4; ∗p ≤ 0.05, ∗∗p ≤ 0.01, one-way ANOVA followed by Tukey multiple comparisons post-test).
See also Figure S6.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions