GC content shapes mRNA storage and decay in human cells.

mRNA translation and decay appear often intimately linked although the rules of this interplay are poorly understood. In this study, we combined our recent P-body transcriptome with transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors, and with available CLIP and related data. This revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA stability: P-bodies contain mostly AU-rich mRNAs, which have a particular codon usage associated with a low protein yield; AU-rich and GC-rich transcripts tend to follow distinct decay pathways; and the targets of sequence-specific RBPs and miRNAs are also biased in terms of GC content. Altogether, these results suggest an integrated view of post-transcriptional control in human cells where most translation regulation is dedicated to inefficiently translated AU-rich mRNAs, whereas control at the level of 5' decay applies to optimally translated GC-rich mRNAs.

Regulatory principles governing the maternal-to-zygotic transition: insights from Drosophila melanogaster.

The onset of metazoan development requires that two terminally differentiated germ cells, a sperm and an oocyte, become reprogrammed to the totipotent embryo, which can subsequently give rise to all the cell types of the adult organism. In nearly all animals, maternal gene products regulate the initial events of embryogenesis while the zygotic genome remains transcriptionally silent. Developmental control is then passed from mother to zygote through a process known as the maternal-to-zygotic transition (MZT). The MZT comprises an intimately connected set of molecular events that mediate degradation of maternally deposited mRNAs and transcriptional activation of the zygotic genome. This essential developmental transition is conserved among metazoans but is perhaps best understood in the fruit fly, Drosophila melanogaster. In this article, we will review our understanding of the events that drive the MZT in Drosophila embryos and highlight parallel mechanisms driving this transition in other animals.

Clearance of Maternal RNAs: Not a Mummy's Embryo Anymore.

Until the zygotic genome is activated, early development relies on the products deposited by the mother. Once the zygotic genome starts to be transcribed, most maternal products are not needed anymore by the developing embryo. This emancipation from the maternal genome occurs during the Zygotic Genome Activation (ZGA). Although the process by which the maternal content is replaced with zygotic products differs from species to species, there is a common theme to all of them: maternal transcripts are actively degraded. Here, a review of how the degradation of maternal RNAs is regulated during early development and discussions on some computational tools that may be of use in this research area are outlined.

m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition.

The maternal-to-zygotic transition (MZT) is one of the most profound and tightly orchestrated processes during the early life of embryos, yet factors that shape the temporal pattern of vertebrate MZT are largely unknown. Here we show that over one-third of zebrafish maternal messenger RNAs (mRNAs) can be N6-methyladenosine (m6A) modified, and the clearance of these maternal mRNAs is facilitated by an m6A-binding protein, Ythdf2. Removal of Ythdf2 in zebrafish embryos decelerates the decay of m6A-modified maternal mRNAs and impedes zygotic genome activation. These embryos fail to initiate timely MZT, undergo cell-cycle pause, and remain developmentally delayed throughout larval life. Our study reveals m6A-dependent RNA decay as a previously unidentified maternally driven mechanism that regulates maternal mRNA clearance during zebrafish MZT, highlighting the critical role of m6A mRNA methylation in transcriptome switching and animal development.

Mature maternal mRNAs are longer than zygotic ones and have complex degradation kinetics in sea urchin.

Early in embryogenesis, maternally deposited transcripts are degraded and new zygotic transcripts are generated during the maternal to zygotic transition. Recent works have shown that early zygotic transcripts are short compared to maternal transcripts, in zebrafish and Drosophila species. The reduced zygotic transcript length was attributed to the short cell cycle in these organisms that prevents the transcription of long primary transcripts (intron delay). Here we study the length of maternal mRNAs and their degradation kinetics in two sea urchin species to further the understanding of maternal gene usage and processing. Early zygotic primary transcripts and mRNAs are shorter than maternal ones in the sea urchin, Strongylocentrotus purpuratus. Yet, while primary transcripts length increases when cell cycle lengthens, typical for intron delay, the relatively short length of zygotic mRNAs is consistent. The enhanced mRNA length is due to significantly longer maternal open reading frames and 3'UTRs compared to the zygotic lengths, a ratio that does not change with developmental time. This implies unique usage of both coding sequences and regulatory information in the maternal stage compared to the zygotic stages. We extracted the half-lifetimes due to maternal and zygotic degradation mechanisms from high-density time course of a set of maternal mRNAs in Paracentrotus lividus. The degradation rates due to maternal and zygotic degradation mechanisms are not correlated, indicating that these mechanisms are independent and relay on different regulatory information. Our studies illuminate specific structural and kinetic properties of sea urchin maternal mRNAs that might be broadly shared by other organisms.

Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation.

Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3'-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.

Positive and negative cis-regulatory elements directing postfertilization maternal mRNA translational control in mouse embryos.

Mechanisms providing for temporally complex patterns of maternal mRNA translation after fertilization are poorly understood. We employed bioinformatics analysis to compare populations of mRNAs enriched specifically on polysomes at the metaphase II (MII) stage oocyte and late one-cell stages and a detailed deletion/truncation series to identify elements that regulate translation. We used the Bag4 3' untranslated region (UTR) as a model. Bioinformatics analysis revealed one conserved motif, subsequently confirmed by functional studies to be a key translation repressor element. The deletion/truncation studies revealed additional regulatory motifs, most notably a strong translation activator element of <30 nt. Analysis of mRNA secondary structure suggests that secondary structure plays a key role in translation repression. Additional bioinformatics analysis of the regulated mRNA population revealed a diverse collection of regulatory motifs found in small numbers of mRNAs, highlighting a high degree of sequence diversity and combinatorial complexity in the overall control of the maternal mRNA population. We conclude that translational control after fertilization is driven primarily by negative regulatory mechanisms opposing strong translational activators, with stage-specific release of the inhibitory influences to permit recruitment. The combination of bioinformatics analysis and deletion/truncation studies provides the necessary approach for dissecting postfertilization translation regulatory mechanisms.

Postmortem stability of RNA isolated from bovine reproductive tissues.

Molecular biology is being increasingly used to address the complex problem of bovine infertility. One common concern shared by many of these studies is the postmortem delay in obtaining reproductive tissues and the effect this may have on RNA dependent studies. To address this concern, bovine ovarian, oviduct and uterine tissue samples, collected over intervals ranging from 0 to 96 h postmortem to freeze storage, were analysed to determine the potential effects on RNA quantity and quality. The analysis showed that total RNA yields were not changed significantly by postmortem interval up to 96 h while 28S ribosomal RNA remained intact up to 24 h postmortem. Specific messenger RNA transcripts encoding beta-actin, GAPDH and transforming growth factor-beta were detected in all tissues up to 96 h postmortem using reverse transcriptase-polymerase chain reaction and Northern analysis indicated no detectable mRNA degradation up to 24 h postmortem. Finally, using poly(A)(+) mRNA isolated from ovarian tissues frozen 2 h postmortem, we constructed corpus luteum and ovarian cortex cDNA libraries containing 7.65x10(4) and 1.9x10(6) primary transformants with average cDNA lengths of 2.3 and 1.6 kb respectively. Taken together, these data show that a postmortem delay of up to 24 h does not significantly affect the yield or quality of RNA prepared from bovine reproductive tissues.