Vedran Franke

A Retrotransposon-Driven Dicer Isoform Directs Endogenous Small Interfering RNA Production in Mouse Oocytes

In mammals, a single Dicer participates in biogenesis of small RNAs in microRNA (miRNA) and RNAi pathways. In mice, endogenous RNAi is highly active in oocytes, but not in somatic cells, which we ascribe here to an oocyte-specific Dicer isoform (Dicer(O)). Dicer(O) lacks the N-terminal DExD helicase domain and has higher cleavage activity than the full-length Dicer in somatic cells (Dicer(S)). Unlike Dicer(S), Dicer(O) efficiently produces small RNAs from long double-stranded (dsRNA) substrates. Expression of the Dicer(O) isoform is driven by an intronic MT-C retrotransposon promoter, deletion of which causes loss of Dicer(O) and female sterility. Oocytes from females lacking the MT-C element show meiotic spindle defects and increased levels of endogenous small interfering RNA (endo-siRNA) targets, phenocopying the maternal Dicer null phenotype. The alternative Dicer isoform, whose phylogenetic origin demonstrates evolutionary plasticity of RNA-silencing pathways, is the main determinant of endogenous RNAi activity in the mouse female germline.

The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3′ processing

Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1‐cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA‐Seq, we have found that transcription in 1‐cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core‐promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1‐cell embryos because of inefficient splicing and 3′ processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1‐cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1‐cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1‐cell embryos.

Using Cloud Computing Infrastructure with CloudBioLinux, CloudMan, and Galaxy

Cloud computing has revolutionized availability and access to computing and storage resources, making it possible to provision a large computational infrastructure with only a few clicks in a Web browser. However, those resources are typically provided in the form of low‐level infrastructure components that need to be procured and configured before use. In this unit, we demonstrate how to utilize cloud computing resources to perform open‐ended bioinformatic analyses, with fully automated management of the underlying cloud infrastructure. By combining three projects, CloudBioLinux, CloudMan, and Galaxy, into a cohesive unit, we have enabled researchers to gain access to more than 100 preconfigured bioinformatics tools and gigabytes of reference genomes on top of the flexible cloud computing infrastructure. The protocol demonstrates how to set up the available infrastructure and how to use the tools via a graphical desktop interface, a parallel command‐line interface, and the Web‐based Galaxy interface. Curr. Protoc. Bioinform. 38:11.9.1‐11.9.20.

Genome-wide analysis of the chromatin composition of histone H2A and H3 variants in mouse embryonic stem cells.

Genome-wide distribution of the majority of H2A and H3 variants (H2A, H2AX, H2AZ, macroH2A, H3.1, H3.2 and H3.3) was simultaneously investigated in mouse embryonic stem cells by chromatin immunoprecipitation sequencing. Around the transcription start site, histone variant distribution differed between genes possessing promoters of high and low CpG density, regardless of their expression levels. In the intergenic regions, regulatory elements were enriched in H2A.Z and H3.3, whereas repeat elements were abundant in H2A and macroH2A, and H3.1, respectively. Analysis of H2A and H3 variant combinations composing nucleosomes revealed that the H2A.Z and H3.3 combinations were present at a higher frequency throughout the genome than the other combinations, suggesting that H2A.Z and H3.3 associate preferentially with each other to comprise the nucleosomes independently of genome region. Finally, we found that chromatin was unstable only in regions where it was enriched in both H2A.Z and H3.3, but strongly quantified stable in regions in which only H3.3 was abundant. Therefore, histone variant composition is an important determinant of chromatin structure, which is associated with specific genomic functions.

Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.

Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development

ABSTRACT The oocyte-to-embryo transition entails genome activation and a dramatic reprogramming of gene expression that is required for continued development. Superimposed on genome activation and reprogramming is development of a transcriptionally repressive state at the level of chromatin structure. Inducing global histone hyperacetylation relieves this repression and histone deacetylases 1 and 2 (HDAC1 and HDAC2) are involved in establishing the repressive state. Because SIN3A is an HDAC1/2-containing complex, we investigated whether it is involved in reprogramming gene expression during the course of genome activation. We find that Sin3a mRNA is recruited during maturation and that inhibiting its recruitment not only inhibits development beyond the 2-cell stage but also compromises the fidelity of reprogramming gene expression. The SIN3A that is synthesized during oocyte maturation reaches a maximum level in the mid-1-cell embryo and is essentially absent by the mid-2-cell stage. Overexpressing SIN3A in 1-cell embryos has no obvious effect on pre- and postimplantation development. These results provide a mechanism by which reprogramming can occur using a maternally inherited transcription machinery, namely, recruitment of mRNAs encoding transcription factors and chromatin remodelers, such as SIN3A.

Long non-coding RNA exchange during the oocyte-to-embryo transition in mice

Abstract The oocyte-to-embryo transition (OET) transforms a differentiated gamete into pluripotent blastomeres. The accompanying maternal-zygotic RNA exchange involves remodeling of the long non-coding RNA (lncRNA) pool. Here, we used next generation sequencing and de novo transcript assembly to define the core population of 1,600 lncRNAs expressed during the OET (lncRNAs). Relative to mRNAs, OET lncRNAs were less expressed and had shorter transcripts, mainly due to fewer exons and shorter 5′ terminal exons. Approximately half of OET lncRNA promoters originated in retrotransposons suggesting their recent emergence. Except for a small group of ubiquitous lncRNAs, maternal and zygotic lncRNAs formed two distinct populations. The bulk of maternal lncRNAs was degraded before the zygotic genome activation. Interestingly, maternal lncRNAs seemed to undergo cytoplasmic polyadenylation observed for dormant mRNAs. We also identified lncRNAs giving rise to trans-acting short interfering RNAs, which represent a novel lncRNA category. Altogether, we defined the core OET lncRNA transcriptome and characterized its remodeling during early development. Our results are consistent with the notion that rapidly evolving lncRNAs constitute signatures of cells-of-origin while a minority plays an active role in control of gene expression across OET. Our data presented here provide an excellent source for further OET lncRNA studies.

Prediction of Interacting Protein Residues Using Sequence and Structure Data

Identifying hotspots responsible for protein interactions with other macromolecules or drugs provides insight into functional aspects of the protein network, and is a pivotal task in systems biology and drug discovery. Here, we present the protocol for the application of a machine-learning method - Random Forest - to prediction of interacting residues in proteins, based on either the structural parameters or the primary sequence alone.