Expression of human ARGONAUTE 2 inhibits endogenous microRNA activity in Arabidopsis

Plant and animal microRNA (miRNA) pathways share many analogous components, the ARGONAUTE (AGO) proteins being foremost among them. We sought to ascertain the degree of functional conservation shared by Homo sapiens ARGONAUTE 2 (HsAGO2) and Arabidopsis thaliana ARGONAUTE 1 (AtAGO1), which are the predominant AGO family members involved with miRNA activity in their respective species. Transgenic Arabidopsis plants expressing HsAGO2 were indistinguishable from counterparts over-expressing AtAGO1, each group exhibiting the morphological and molecular hallmarks of miRNA-pathway loss-of-function alleles. However, unlike AtAGO1, HsAGO2 was unable to rescue the ago1-27 allele. We conclude that, despite the evolutionary gulf between them, HsAGO2 is likely capable of interacting with some component/s of the Arabidopsis miRNA pathway, thereby perturbing its operation, although differences have arisen such that HsAGO2 alone is insufficient to confer efficient silencing of miRNA targets in planta

Target RNA Secondary Structure Is a Major Determinant of miR159 Efficacy

In plants, microRNA (miRNA)-target complementarity has long been considered the predominant factor determining the silencing outcome of the miRNA-target interaction, although the efficacy of such interactions have rarely been appraised in plants. Here, we perform in planta silencing efficacy assays on seven Arabidopsis MYB genes, all of which contain conserved miR159-binding sites of analogous complementarity. These genes were found to be differentially silenced by miR159; MYB81, MYB97, MYB101, MYB104, and DUO1 were all poorly silenced, whereas MYB33 and MYB65 were strongly silenced. Curiously, this is consistent with previous genetic analysis defining MYB33 and MYB65 as the major functional targets of miR159. Neither the free energy of miR159-target complementarity, nor miRNA binding site accessibility, as determined by flanking region AU content, could fully explain the discrepancy of miR159 silencing efficacy. Instead, we found that MYB33 and MYB65 were both predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site. The structure is composed of two stem-loops (SLs) that are predicted to form in MYB33/65 homologs of species as evolutionary distant as gymnosperms. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing complementary mutations to restore the SLs, but not the sequence, restored strong miR159-mediated silencing. Therefore, it appears that this RNA secondary structure demarcates MYB33/65 as sensitive targets of miR159, which underpins the narrow functional specificity of Arabidopsis miR159. MicroRNAs (miRNAs) are small 20- to 24-nucleotide (nt) RNAs that guide the RNA-Induced Silencing Complex to target mRNAs and mediate their silencing through a combination of transcript degradation and translational repression (Axtell, 2013). In plants, miRNAs have been shown to be involved in a multitude of critical developmental events and stress responses, and are often referred to as master regulators of gene expression. Central to understanding miRNA function has been identifying their target mRNAs (Sun et al., 2014). In plants, it is clear that high sequence complementarity between a miRNA and its target mRNA is compulsory for a miRNA-target interaction (Mallory et al., 2004; Schwab et al., 2005; Addo-Quaye et al., 2008; German et al., 2008), with most experimentally validated miRNA-target pairs having very few mismatches (Schwab et al., 2005; Liu et al., 2014). Consequently, miRNA-target complementarity has been the cornerstone of plant miRNA biology, determining miRNA target prediction (Dai and Zhao, 2011), the design of artificial miRNAs (amiRNAs; Schwab et al., 2005), the design of artificial miRNA decoys such as target MIMICs (Todesco et al., 2010), or the identification of endogenous target MIMICs (Karakülah et al., 2016). However, bioinformatic prediction of target genes often fails to accurately predict functionally relevant targets, where from numerous predicted targets, only a select few appear functionally significant (for review, see Li et al., 2014a). Likewise, it has been reported that amiRNAs with high complementarity to their intended targets perform with considerable variability in plants (Li et al., 2013; Deveson et al., 2013). Finally, different miRNA decoys that contain identical miRNA binding sites work with widely varying efficacies (Reichel et al., 2015). These and other observations argue that miRNA-target interaction is not simply a product of complementarity, but additional factors are required for functional miRNA-target interactions (Wang et al., 2015). In animals, it has long been known that the contextual sequence features in which a miRNA-binding site resides can strongly impact silencing. For example, miRNA-binding site accessibility was shown to be important, where introduction of mutations to decrease predicted accessibility disrupted efficient regulation, with impacts being as strong as mutations within the binding site itself (Kertesz et al., 2007). Furthermore, it has been shown that for certain animal miRNA-target interactions, strong regulation only occurs when the binding sites are within specific sequence arrangements or contexts (Didiano and Hobert, 2006; Vella et al., 2004). Other factors impacting the efficiency of regulation include RNA-binding proteins (RBPs), which can either attenuate or facilitate the access of the miRNA to its binding site (Kedde et al., 2010). Factors such as these have been studied less in plants, but evidence is accumulating that sequence context of miRNA binding sites may also be important in plants. First, Gu et al. (2012) found a synonymous codon bias favoring AU-richness, and hence reduced RNA secondary structure, around predicted miRNA target sites in several plant species. Second, Li et al. (2012) have found that miRNA binding sites in Arabidopsis (Arabidopsis thaliana) are generally less structured than their flanking regions, indicating a preference for high accessibility. Indeed, Fei et al. (2015) found that target site accessibility may explain select regulation of only a few targets from a large number of predicted target genes. Therefore, it would be of interest to functionally test these potential factors on miRNA-target interactions. In plants, the Arabidopsis miR159 family has been extensively studied as a model for plant miRNA-mediated gene regulation (Palatnik et al., 2003, 2007; Allen et al., 2007, 2010). The family has two major isoforms, miR159a and miR159b, which are strongly expressed throughout Arabidopsis (Palatnik et al., 2007; Li et al., 2016). Such expression is consistent with a loss-of-function mir159ab double mutant that displays strong pleiotropic developmental defects. In Arabidopsis, miR159 is bioinformatically predicted to regulate more than 20 targets, including eight genes encoding conserved R2R3 domain MYB transcription factors (Palatnik et al., 2007). Despite this, genetic analysis revealed that miR159-mediated regulation of only two of the predicted target genes, MYB33 and MYB65, account for the developmental defects of mir159ab, as all defects are suppressed in a myb33.myb65.mir159ab quadruple mutant (Allen et al., 2007). This defined the functional specificity of Arabidopsis miR159 being restricted to MYB33 and MYB65, but also raised the question of the functional significance of miR159-mediated regulation of the additional bioinformatically predicted targets, including those that have a strongly conserved miR159 binding site (Allen et al., 2007, 2010). Curiously, this narrower functional specificity as defined by genetics has also been found in other plant and animal miRNA systems, suggesting the functional scope of miRNA-mediated silencing is narrower than generally assumed (Seitz, 2009; Li et al., 2014a). For miR159, it is likely that multiple factors contribute to this apparent narrow functional specificity, including nonoverlapping transcriptional domains of the miR159 and MYB target genes (Allen et al., 2007), whether regulation of other targets is important under certain untested growth conditions or certain miR159 isoforms have become obsolete (Allen at al., 2010). However, one untested hypothesis is that MYB33 and MYB65 are more sensitive to miR159 regulation than the other MYB target genes. Recently, we have shown that factors beyond complementary govern the efficacy of the miR159-MYB33 silencing outcome (Li et al., 2014b). This not only included the miR159:MYB33 transcript stoichiometry, but also the sequence context of the miR159 binding site in MYB33. We showed that mutation of nts that immediately flank the miR159-binding site attenuated silencing to a similar extent to mutating nts within the binding site itself (Li et al., 2014b). This is further evidence that sequence complementarity alone does not guarantee strong miRNA regulation and that additional factor(s) are at play impacting miRNA-mediated regulation in plants. Here, by carrying out in planta miR159 efficacy assays, we show that MYB33 and MYB65 are indeed much more sensitive to miR159 regulation than the other MYB genes with conserved miR159 binding sites. Neither flanking AU content nor predicted accessibility of the miR159 binding site underlie this sensitivity. Rather, MYB33 and MYB65 are shown to share a predicted RNA secondary structure consisting of two stem-loops that partially overlap with the miR159 binding site. Structure-function analysis demonstrates that both these stem-loops are required for efficient miR159 mediated silencing of MYB33. We hypothesize that having strong RNA stem-loops adjacent to a miRNA binding site may facilitate accessibility of the binding site to the miRNA, which in turn promotes efficient silencing of the target gene.This work was supported by a Research School of Biology International student Ph.D. scholarship to Z.Z., an International ANU Ph.D. scholarship to M.R., and an Australian Research Council Discovery Grant (No. DP110103493) to A.A.M

Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing

More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care

Universal Alternative Splicing of Noncoding Exons

The human transcriptome is so large, diverse, and dynamic that, even after a decade of investigation by RNA sequencing (RNA-seq), we have yet to resolve its true dimensions. RNA-seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed targeted single-molecule and short-read RNA-seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome, identifying a fundamental distinction between protein-coding and noncoding gene content: almost every noncoding exon undergoes alternative splicing, producing a seemingly limitless variety of isoforms. Analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse, yet human splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution

A 1000-year-old case of Klinefelter's syndrome diagnosed by integrating morphology, osteology, and genetics

We thank the Municipality of Bragança, the University of Coimbra, the University of Adelaide, the Max Planck Society, and the Calouste Gulbenkian Foundation for the support provided. ST is supported by the Fundação para a Ciência e a Tecnologia (SFRH/BD/116363/2016). BL (FT170100448) and JCT (DE210101235) are supported by the Australian Research Council. ABR is supported by the European Research Council (771234-PALEoRIDER)

Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications. </p

Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis

Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing (WGS) data from nearly 10,000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n=6 from 5 families), AAGGC (n=2 from 1 family), AGAGG (n=1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, here we show a pathogenic role for large AAAGG repeat configuration expansions (n=5). Long read sequencing was used to fully characterise the entire repeat sequence and revealed a pure AGGGC expansion in six patients, whereas the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs seem to have arisen from a common haplotype and are predicted to form highly stable G quadruplexes, which have been previously demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions, since the AAAGG motif when very large (>500 repeats) or in the presence of AAGGG interruptions. Accurate sizing and full sequencing of the satellite repeat with long read is recommended in clinically selected cases, in order to achieve an accurate molecular diagnosis and counsel patients and their families

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2

Publisher Copyright: © 2021 O'Toole Á et al.Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.Peer reviewe

MicroRNAs with analogous target complementarities perform with highly variable efficacies in Arabidopsis

In plants, the silencing efficacy of microRNAs (miRNAs) is thought to be predominantly determined by the degree of complementarity to their target genes. Here, silencing efficacy was determined for Arabidopsis miR159 and four artificial miRNAs (amiRNAs)

Three (largely unrelated) experiments in the age of next-generation sequencing

Next generation sequencing (NGS) enables researchers to identify instances of genetic variation and measure gene expression in an unbiased, global fashion. In this thesis I describe three experiments that apply NGS in quite distinct contexts. Together these illustrate the strengths and broad utility of this revolutionary technology.Experiment 1. I have developed a set of synthetic DNA standards – termed sequins – that emulate human genetic features and constitute qualitative and quantitative spike- in controls for NGS. I have used this approach to represent common and clinically relevant genetic variation, ranging from single nucleotide variants to large structural rearrangements. Here I describe the design and validation of sequin controls, and demonstrate their capacity to measure and mitigate biases during NGS analysis.Experiment 2. In many vertebrates sex is determined by external environmental cues rather than by sex chromosomes. In reptiles, for instance, temperature-dependent sex determination (TSD) is common. I have used the Australian central bearded dragon, in which chromosomal sex determination is overridden at high temperatures to produce sex-reversed female offspring, as a unique model to identify TSD-specific features of the transcriptome. Here I show that an intron is retained in mature transcripts from each of two Jumonji-family chromatin modifier genes, JARID2 and JMJD3, in female dragons that have been sex-reversed by temperature, but not in normal chromosomal females or males. I also observed sex-associated differential retention of the equivalent introns expressed in alligators and turtles, indicative of a reptile-wide mechanism that may control TSD.Experiment 3. The human transcriptome is so large, diverse and dynamic that, even after a decade of investigation by RNA sequencing (RNA-Seq), we are yet to resolve its true dimensions. I have performed targeted single-molecule and short-read RNA- Seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. This analysis identifies a fundamental distinction between the architecture of protein-coding and noncoding gene content. Unlike their coding counterparts, noncoding exons undergo universal alternative splicing to produce a seemingly limitless variety of isoforms. I propose that noncoding exons are functionally modular, with combinatorial alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution