A small RNA that cooperatively senses two stacked metabolites in one pocket for gene control

Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ1-sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development.

The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA

The control of messenger RNA (mRNA) translation has been increasingly recognized as a key regulatory step for gene control, but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf). In this study, we focused on the Ogura CMS system in rapeseed and showed that reversion to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific translation inhibition of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

Transcriptional enhancers and their communication with gene promoters

Transcriptional enhancers play a key role in the initiation and maintenance of gene expression programmes, particularly in metazoa. How these elements control their target genes in the right place and time is one of the most pertinent questions in functional genomics, with wide implications for most areas of biology. Here, we synthesise classic and recent evidence on the regulatory logic of enhancers, including the principles of enhancer organisation, factors that facilitate and delimit enhancer–promoter communication, and the joint effects of multiple enhancers. We show how modern approaches building on classic insights have begun to unravel the complexity of enhancer–promoter relationships, paving the way towards a quantitative understanding of gene control.

Transcription-coupled and epigenome-encoded mechanisms direct H3K4 methylation

Mono-, di-, and trimethylation of histone H3 lysine 4 (H3K4me1/2/3) are associated with transcription, yet it remains controversial whether H3K4me1/2/3 promote or result from transcription. Our previous characterizations of Arabidopsis H3K4 demethylases suggest roles for H3K4me1 in transcription. However, the control of H3K4me1 remains unexplored in Arabidopsis, in which no methylase for H3K4me1 has been identified. Here, we identified three Arabidopsis methylases that direct H3K4me1. Analyses of their genome-wide localization using ChIP-seq and machine learning revealed that one of the enzymes cooperates with the transcription machinery, while the other two are associated with specific histone modifications and DNA sequences. Importantly, these two types of localization patterns are also found for the other H3K4 methylases in Arabidopsis and mice. These results suggest that H3K4me1/2/3 are established and maintained via interplay with transcription as well as inputs from other chromatin features, presumably enabling elaborate gene control.

RNA-basierte Regulation der Genexpression: künstliche Genschalter

RNA-based gene control mechanisms pose an elegant and straightforward way to switch on, off, or fine-tune transgene expression without the need for expressing regulatory proteins. A small molecule effector binds directly to a ligand-binding aptamer RNA structure and thereby modulates expression of an associated target gene. We established genetic switches based on regulation of self-cleaving ribozymes and polyadenylation that allow for control of transgene expression in bacteria, yeast, human cell lines and Caenorhabditis elegans in a robust and dose-dependent manner.

The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA

The control of mRNA translation has been increasingly recognized as a key regulatory step for gene control but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf) genes. In the study, we focused on the Ogura CMS system in rapeseed and showed that the suppression to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific inhibition of the translation of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

Enhanced Cas12a multi-gene regulation using a CRISPR array separator

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment a CRISPR separator. Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

Nanobody-mediated control of gene expression and epigenetic memory

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

Identifying new variation at the J locus, previously identified as e6, in long juvenile ‘Paranagoiana’ soybean

Key message A previously identified soybean maturity locus, E6, is discovered to be J, with the long juvenile allele in Paranagoiana now deemed j−x. Abstract Soybean grown at latitudes of ~20° or lower can produce lower grain yields due to the short days. This limitation can be overcome by using the long juvenile trait (LJ) which delays flowering under short day conditions. Two LJ loci have been mapped to the same location on Gm04, J and E6. The objective of this research was to investigate the e6 allele in ‘Paranagoiana’ and determine if E6 and J are the same locus or linked loci. KASP markers showed that e6 lines did not have the j−1 allele of LJ PI 159925. A population fixed for E1 but segregating for E6, with e6 introgressed from Paranagoiana, showed single gene control for flowering and maturity under short days. Sequencing Glyma.04G050200, the J gene, with long amplification Taq found that the e6 line ‘Paranagoiana’ contains a Ty1-copia retrotransposon of ~10,000 bp, inserted within exon 4. PCR amplification of the cDNA of Glyma.04G050200 also showed differences between the mRNA sequences (presence of insertion in j−x). Hence, we conclude that the loci E6 and J are one locus and deem this new variation found in Paranagoiana as j−x.