Limits...
An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation

View Article: PubMed Central - PubMed

ABSTRACT

Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis.

No MeSH data available.


Specific cis-Acting Elements and Elevated Transcription Rate Feature Meiotic IRTs(A) Graphic representation of retained introns location within gene body.(B–D) Box plots representing comparison between retained introns and other introns for their length (B), GC content (C), and splice-site strength (D). Whiskers indicate 1.5 interquartile range. p Values indicate a significant difference between means of the two populations (Welch's t test).(E) Box plots showing distribution of FPKM values for IR and other expressed genes in spermatocytes (sp.cytes) and spermatids (sp.tids). p Values indicate a significant difference between means of the two groups in the two cell types (Welch's t test).(F) Bar graph showing percentages of intron-retaining and other expressed genes enriched for indicated histone marks in sp.cytes. p Values indicate a significant difference in the enrichment for different histone marks between the two groups (χ2 test).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5392497&req=5

fig4: Specific cis-Acting Elements and Elevated Transcription Rate Feature Meiotic IRTs(A) Graphic representation of retained introns location within gene body.(B–D) Box plots representing comparison between retained introns and other introns for their length (B), GC content (C), and splice-site strength (D). Whiskers indicate 1.5 interquartile range. p Values indicate a significant difference between means of the two populations (Welch's t test).(E) Box plots showing distribution of FPKM values for IR and other expressed genes in spermatocytes (sp.cytes) and spermatids (sp.tids). p Values indicate a significant difference between means of the two groups in the two cell types (Welch's t test).(F) Bar graph showing percentages of intron-retaining and other expressed genes enriched for indicated histone marks in sp.cytes. p Values indicate a significant difference in the enrichment for different histone marks between the two groups (χ2 test).

Mentions: To gain insight into the nature of the IRTs enriched in meiosis, we explored whether retained introns displayed specific structural features that may distinguish them from properly spliced introns. Bioinformatics analysis documented that retained introns are preferentially localized toward the 3′ end of genes (Figure 4A). Similarly to what was previously reported for other introns affected by developmentally or physiologically regulated retention (Braunschweig et al., 2014, Boutz et al., 2015), meiotically retained introns are significantly shorter in length and display a higher GC content (Figures 4B and 4C). Furthermore, they are flanked by weaker 5′ and 3′ splice sites when compared with all other introns (Figure 4D). These results suggest that meiotically retained introns represent a distinct class characterized by specific features, which might contribute to the mechanism(s) underlying their retention. Intriguingly, by evaluating the mean FPKM (fragments per kilobase of transcript per million mapped reads) value as estimate of the gene expression levels (Trapnell et al., 2010), we observed significantly higher expression in the group of IR genes compared with all the other transcribed genes (Figure 4E). Moreover, comparison of our dataset with chromatin immunoprecipitation sequencing (ChIP-seq) analyses of histone marks in spermatocytes available from the GEO database (GSE49624; Hammoud et al., 2014) highlighted a significant enrichment of epigenetic marks of active transcription (i.e., acetylated H3K27, trimethylated H3K4) in the 5′ region of IR genes with respect to all other genes, whereas repressive marks (i.e., trimethylated H3K27) were not affected (Figure 4F). These results suggest that the high transcriptional level of IR genes combined with the specific features of their sequence negatively selects retained introns from recognition and excision by the spliceosome in meiosis.


An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation
Specific cis-Acting Elements and Elevated Transcription Rate Feature Meiotic IRTs(A) Graphic representation of retained introns location within gene body.(B–D) Box plots representing comparison between retained introns and other introns for their length (B), GC content (C), and splice-site strength (D). Whiskers indicate 1.5 interquartile range. p Values indicate a significant difference between means of the two populations (Welch's t test).(E) Box plots showing distribution of FPKM values for IR and other expressed genes in spermatocytes (sp.cytes) and spermatids (sp.tids). p Values indicate a significant difference between means of the two groups in the two cell types (Welch's t test).(F) Bar graph showing percentages of intron-retaining and other expressed genes enriched for indicated histone marks in sp.cytes. p Values indicate a significant difference in the enrichment for different histone marks between the two groups (χ2 test).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5392497&req=5

fig4: Specific cis-Acting Elements and Elevated Transcription Rate Feature Meiotic IRTs(A) Graphic representation of retained introns location within gene body.(B–D) Box plots representing comparison between retained introns and other introns for their length (B), GC content (C), and splice-site strength (D). Whiskers indicate 1.5 interquartile range. p Values indicate a significant difference between means of the two populations (Welch's t test).(E) Box plots showing distribution of FPKM values for IR and other expressed genes in spermatocytes (sp.cytes) and spermatids (sp.tids). p Values indicate a significant difference between means of the two groups in the two cell types (Welch's t test).(F) Bar graph showing percentages of intron-retaining and other expressed genes enriched for indicated histone marks in sp.cytes. p Values indicate a significant difference in the enrichment for different histone marks between the two groups (χ2 test).
Mentions: To gain insight into the nature of the IRTs enriched in meiosis, we explored whether retained introns displayed specific structural features that may distinguish them from properly spliced introns. Bioinformatics analysis documented that retained introns are preferentially localized toward the 3′ end of genes (Figure 4A). Similarly to what was previously reported for other introns affected by developmentally or physiologically regulated retention (Braunschweig et al., 2014, Boutz et al., 2015), meiotically retained introns are significantly shorter in length and display a higher GC content (Figures 4B and 4C). Furthermore, they are flanked by weaker 5′ and 3′ splice sites when compared with all other introns (Figure 4D). These results suggest that meiotically retained introns represent a distinct class characterized by specific features, which might contribute to the mechanism(s) underlying their retention. Intriguingly, by evaluating the mean FPKM (fragments per kilobase of transcript per million mapped reads) value as estimate of the gene expression levels (Trapnell et al., 2010), we observed significantly higher expression in the group of IR genes compared with all the other transcribed genes (Figure 4E). Moreover, comparison of our dataset with chromatin immunoprecipitation sequencing (ChIP-seq) analyses of histone marks in spermatocytes available from the GEO database (GSE49624; Hammoud et al., 2014) highlighted a significant enrichment of epigenetic marks of active transcription (i.e., acetylated H3K27, trimethylated H3K4) in the 5′ region of IR genes with respect to all other genes, whereas repressive marks (i.e., trimethylated H3K27) were not affected (Figure 4F). These results suggest that the high transcriptional level of IR genes combined with the specific features of their sequence negatively selects retained introns from recognition and excision by the spliceosome in meiosis.

View Article: PubMed Central - PubMed

ABSTRACT

Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis.

No MeSH data available.