Smad-mediated regulation of microRNA biosynthesis

AbstractmicroRNAs (miRNAs) are small non-coding RNAs conserved in metazoans. Depletion of miRNAs results in embryonic lethality, suggesting they are essential for embryogenesis. Similarly, pathways induced by growth factors of the transforming growth factor β (TGF-β) superfamily control cell growth, differentiation, and development. Recently Smad proteins, the signal transducers of the TGF-β pathway, were found to regulate miRNA expression, which, in turn, affects expression of numerous proteins. Smads modulate miRNA expression through both transcriptional and post-transcriptional mechanisms illustrating the complexity of gene regulation by TGF-β. In this chapter we summarize the current knowledge of mechanisms underlying Smad-mediated regulation of miRNA biogenesis

Zcchc11 Uridylates Mature miRNAs to Enhance Neonatal IGF-1 Expression, Growth, and Survival

The Zcchc11 enzyme is implicated in microRNA (miRNA) regulation. It can uridylate let-7 precursors to decrease quantities of the mature miRNA in embryonic stem cell lines, suggested to mediate stem cell maintenance. It can uridylate mature miR-26 to relieve silencing activity without impacting miRNA content in cancer cell lines, suggested to mediate cytokine and growth factor expression. Broader roles of Zcchc11 in shaping or remodeling the miRNome or in directing biological or physiological processes remain entirely speculative. We generated Zcchc11-deficient mice to address these knowledge gaps. Zcchc11 deficiency had no impact on embryogenesis or fetal development, but it significantly decreased survival and growth immediately following birth, indicating a role for this enzyme in early postnatal fitness. Deep sequencing of small RNAs from neonatal livers revealed roles of this enzyme in miRNA sequence diversity. Zcchc11 deficiency diminished the lengths and terminal uridine frequencies for diverse mature miRNAs, but it had no influence on the quantities of any miRNAs. The expression of IGF-1, a liver-derived protein essential to early growth and survival, was enhanced by Zcchc11 expression in vitro, and miRNA silencing of IGF-1 was alleviated by uridylation events observed to be Zcchc11-dependent in the neonatal liver. In neonatal mice, Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and IGF-1 protein in the blood. We conclude that the Zcchc11-mediated terminal uridylation of mature miRNAs is pervasive and physiologically significant, especially important in the neonatal period for fostering IGF-1 expression and enhancing postnatal growth and survival. We propose that the miRNA 3′ terminus is a regulatory node upon which multiple enzymes converge to direct silencing activity and tune gene expression

Regulation of miRNA biogenesis as an integrated component of growth factor signaling.

Transcriptional control of microRNAs (miRNA) by cell signaling pathways, especially in the context of growth factor regulation, is a widely recognized phenomenon with broad-reaching implications. However, several recent studies indicate that not just transcription, but also processing of miRNAs is subject to regulation as part of an integrated physiological response to various stimuli and environmental changes. The canonical miRNA biogenesis pathway; sequential steps of nucleolytic cleavage by the RNase III enzymes Drosha and Dicer, are emerging regulatory hubs for the modulation of miRNA expression as part of both physiological and pathological responses. In this article we use well-characterized growth-factor signaling pathways such as transforming growth factor-β (TGF-β), Protein Kinase B (PKB, also known as Akt) and extracellular-signal-regulated kinase (ERK) to illustrate how basic cell signaling pathways modulate the activities of these components of the miRNA biogenesis pathway to achieve optimal miRNA expression patterns

Regulation of miRNA biogenesis as an integrated component of growth factor signaling.

Transcriptional control of microRNAs (miRNA) by cell signaling pathways, especially in the context of growth factor regulation, is a widely recognized phenomenon with broad-reaching implications. However, several recent studies indicate that not just transcription, but also processing of miRNAs is subject to regulation as part of an integrated physiological response to various stimuli and environmental changes. The canonical miRNA biogenesis pathway; sequential steps of nucleolytic cleavage by the RNase III enzymes Drosha and Dicer, are emerging regulatory hubs for the modulation of miRNA expression as part of both physiological and pathological responses. In this article we use well-characterized growth-factor signaling pathways such as transforming growth factor-β (TGF-β), Protein Kinase B (PKB, also known as Akt) and extracellular-signal-regulated kinase (ERK) to illustrate how basic cell signaling pathways modulate the activities of these components of the miRNA biogenesis pathway to achieve optimal miRNA expression patterns

Zcchc11 deficiency decreases IGF-1 expression <i>in vivo</i>.

<p>(A) IGF-1 mRNA, measured by qRT-PCR, was decreased by Zcchc11 deficiency in 8-day-old livers. *p<0.05 <i>vs</i> Zcchc11^+/+ by paired student's t-test. (B) Growth hormone (GH) in serum, measured by ELISA, was unaffected by genotype in 8-day-old mice, based on no significant difference using student's t-test. (C) STAT5 content and phosphorylation, as detected by immunoblot, were unaffected by Zcchc11 deficiency in the 8-day-old liver. (D) Growth factor PCR array of the livers from 8-day-old mice revealed IGF-1 to be the only transcript in this set to be substantially expressed and diminished by Zcchc11 deficiency. Numerical values for each genotype indicate dCT of transcript expression normalized against the mean expression of three separate housekeeping genes. The “fold” column represents the fold-change expression in Zcchc11^−/− compared to Zcchc11^+/+ livers. (E) Histone H3 content, as detected by immunoblot, was unaffected by Zcchc11 deficiency in the 8-day-old liver. (F) IGF-1 protein was decreased in the serum of 8-day-old Zcchc11^−/− mice. *p<0.05 <i>vs</i> Zcchc11^+/+ by student's t-test.</p

Zcchc11 enhances growth and fitness through the perinatal period.

<p>(A) Immunoblots of organs from C57BL/6 and Zcchc11^−/− mice show deletion of Zcchc11 protein. (B) Fraction of homozygous Zcchc11-deficient offspring from Zcchc11^+/− parents at day E14, P1, P8 and P21, indicating decreased survival by day 8. *p<0.05 <i>vs.</i> 0.25 by Chi-squared test, N as indicated. (C) Body weights of Zcchc11^+/+ and Zcchc11^−/− littermates at day 1 and 8 (*p<0.05), showing poor growth in mutants. (D) Let-7 content in primary embryonic stem cell (ESC) cultures that were wild type (+/+), heterozygous (+/−), or deficient (−/−) in Zcchc11 expression, showing no significant effects of genotype (by two-way ANOVA). (E) Proportion of organ weight to body weight in 8-day-old C57BL/6 and Zcchc11^−/− mice, showing no difference between genotypes across tissues and suggesting a system-wide growth defect rather than organ-specific effects. (F) Age-dependent expression of Zcchc11, as shown by immunoblots of tissues from C57BL/6 mice at 2 days, 2 weeks, and 10 weeks of age, reveals strongest expression in most organs at young ages. GAPDH is provided as a loading control.</p

Zcchc11 stabilizes the IGF-1 3′ UTR.

<p>(A) Northern blotting was used to identify the predominant IGF-1 isoform expressed in the livers of wild type and Zcchc11-deficient mice at 8 days old. (B) The 3′ UTR from this isoform, cloned onto the end of a firefly luciferase reporter and co-transfected, along with a <i>Renilla</i> Luciferase containing a minimal promoter for normalization, decreased reporter expression in H1299 cells. (C) These same constructs were transfected in H1299 cells along with plasmids encoding EGFP, Zcchc11, catalytically inactive Zcchc11 (DADA), or the N-terminal half of Zcchc11. The full-length Zcchc11 increased expression of the IGF-1 3′ UTR reporter, which was significantly inhibited by selective mutation of the catalytic domain or complete deletion of the C terminal half. *p<0.05 <i>vs.</i> EGFP <i>†</i>p<0.05 <i>vs</i> Zcchc11 by one-way ANOVA.</p

Zcchc11 is essential for the uridylation of miRNAs.

<p>The miRNA libraries from Zcchc11-defficent and wild type livers at 8 days of age were analyzed for differences in sequence diversity. (A) Histogram of the number of sequences of a given read length across the libraries, showing fewer 23 nucleotide-long reads in the mutant samples. *p<0.05 by two-way ANOVA with Bonferroni <i>post hoc</i> test. (B) End modifications, identified as any nucleotide one base beyond the length of the miRNA published in miRBase, were quantified for each library. Most miRNAs ending in uridine in the control mice did so less frequently in Zcchc11-deficient mice. The mean of the fold change in the percent of sequences adenylated or uridylated between the knockout and wild type were graphed as waterfall plots, with every bar representing the geometric mean of (percent of miR-X sequences modified in the mutants)/(percent of miR-X sequences modified in the WT) for the three separate libraries. Only miRNAs with >10,000 reads and >0.1% sequences end-modified in the wild type libraries were included. Data were expressed as fold-change in Zcchc11^−/− mice compared to Zcchc11^+/+ mice. Light grey bars indicate miRNAs with a seed sequence complementary to a portion of the IGF-1 3′ UTR. (C) Average fold change across all miRNA species shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003105#pgen-1003105-g003" target="_blank">Figure 3B</a>, showing that uridines but not adenines were less frequent at miRNA termini due to Zcchc11 deficiency. Error bars indicate 95% c.i. *p<0.05 <i>vs.</i> 0 by student's t-test. (D) Immunoblots for the indicated non-canonical poly(A) polymerases in tissue homogenates prepared from the livers of 8 day-old mice, showing no effect of genotype. (E) qRT-PCR for Zcchc6 expression in the livers of 8 day-old mice, showing no effect of genotype.</p