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Key message Maize Outer cell layer 4 (ocl4) encodes an HD-ZIP IV transcription factor required for robust male fertility and 21-nt phasiRNA biogenesis . ocl4 fertility is favored in warm conditions, and phasiRNAs are partially restored . Environment-sensitive male-sterile plants have been described before and can result from different molecular mechanisms and biological processes, but putative environment-conditioned, transgenerational rescue of their male fertility is a rather new mystery. Here, we report a derivative line of the male-sterile outer cell layer 4 ( ocl4 ) mutant of maize, in which fertility was restored and perpetuated over several generations. Conditioned fertile ocl4 anthers exhibit the anatomical abnormality of a partially duplicated endothecial layer, just like their sterile counterparts. We profiled the dynamics of phased, small interfering RNAs (phasiRNAs) during pre-meiotic development in fully sterile and various grades of semi-fertile ocl4 anthers . The conditioned fertile anthers accumulated significantly higher 21-nt phasiRNAs compared to ocl4 sterile samples, suggesting a partial restoration of phasiRNAs in conditioned fertility. We found that the biogenesis of 21-nt phasiRNAs is largely dependent on Ocl4 at three key steps: (1) production of PHAS precursor transcripts, (2) expression of miR2118 that modulates precursor processing, and (3) accumulation of 21-nt phasiRNAs.

Background The remarkable growth of genome-wide association studies (GWAS) has created a critical need to experimentally validate the disease-associated variants, 90% of which involve non-coding variants. Methods To determine how the field is addressing this urgent need, we performed a comprehensive literature review identifying 36,676 articles. These were reduced to 1454 articles through a set of filters using natural language processing and ontology-based text-mining. This was followed by manual curation and cross-referencing against the GWAS catalog, yielding a final set of 286 articles. Results We identified 309 experimentally validated non-coding GWAS variants, regulating 252 genes across 130 human disease traits. These variants covered a variety of regulatory mechanisms. Interestingly, 70% (215/309) acted through cis-regulatory elements, with the remaining through promoters (22%, 70/309) or non-coding RNAs (8%, 24/309). Several validation approaches were utilized in these studies, including gene expression (n = 272), transcription factor binding (n = 175), reporter assays (n = 171), in vivo models (n = 104), genome editing (n = 96) and chromatin interaction (n = 33). Conclusions This review of the literature is the first to systematically evaluate the status and the landscape of experimentation being used to validate non-coding GWAS-identified variants. Our results clearly underscore the multifaceted approach needed for experimental validation, have practical implications on variant prioritization and considerations of target gene nomination. While the field has a long way to go to validate the thousands of GWAS associations, we show that progress is being made and provide exemplars of validation studies covering a wide variety of mechanisms, target genes, and disease areas.

Post-transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield noncoding RNAs that are processed into 21- and 24-nucleotide (nt) phased small interfering RNAs (phasiRNAs); these are triggered by miR2118 and miR2275. We characterized these ‘reproductive phasiRNAs’ from rice (Oryza sativa) panicles and anthers across seven developmental stages. Our computational analysis identified characteristics of the 21-nt reproductive phasiRNAs that impact their biogenesis, stability, and potential functions. We demonstrate that 21-nt reproductive phasiRNAs can function in cis to target their own precursors. We observed evidence of this cis regulatory activity in both rice and maize (Zea mays). We validated this activity with evidence of cleavage and a resulting shift in the pattern of phasiRNA production. We characterize biases in phasiRNA biogenesis, demonstrating that the Pol II-derived ‘top’ strand phasiRNAs are consistently higher in abundance than the bottom strand. The first phasiRNA from each precursor overlaps the miR2118 target site, and this impacts phasiRNA accumulation or stability, evident in the weak accumulation of this phasiRNA position. Additional influences on this first phasiRNA duplex include the sequence composition and length, and we show that these factors impact Argonaute loading.

miR-137 plays critical roles in the nervous system and tumor development; an increase in its expression is required for neuronal differentiation while its reduction is implicated in gliomagenesis. To evaluate the potential of miR-137 in glioblastoma therapy, we conducted genome-wide target mapping in glioblastoma cells by measuring the level of association between PABP and mRNAs in cells transfected with miR-137 mimics vs. controls via RIPSeq. Impact on mRNA levels was also measured by RNASeq. By combining the results of both experimental approaches, 1468 genes were found to be negatively impacted by miR-137--among them, 595 (40%) contain miR-137 predicted sites. The most relevant targets include oncogenic proteins and key players in neurogenesis like c-KIT, YBX1, AKT2, CDC42, CDK6 and TGFβ2. Interestingly, we observed that several identified miR-137 targets are also predicted to be regulated by miR-124, miR-128 and miR-7, which are equally implicated in neuronal differentiation and gliomagenesis. We suggest that the concomitant increase of these four miRNAs in neuronal stem cells or their repression in tumor cells could produce a robust regulatory effect with major consequences to neuronal differentiation and tumorigenesis.

Post‐transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield noncoding RNA s that are processed into 21‐ and 24‐nucleotide (nt) phased small interfering RNA s (phasi RNA s); these are triggered by miR2118 and miR2275. We characterized these ‘reproductive phasi RNA s’ from rice ( Oryza sativa ) panicles and anthers across seven developmental stages. Our computational analysis identified characteristics of the 21‐nt reproductive phasi RNA s that impact their biogenesis, stability, and potential functions. We demonstrate that 21‐nt reproductive phasi RNA s can function in cis to target their own precursors. We observed evidence of this cis regulatory activity in both rice and maize ( Zea mays ). We validated this activity with evidence of cleavage and a resulting shift in the pattern of phasi RNA production. We characterize biases in phasi RNA biogenesis, demonstrating that the Pol  II ‐derived ‘top’ strand phasi RNA s are consistently higher in abundance than the bottom strand. The first phasi RNA from each precursor overlaps the miR2118 target site, and this impacts phasi RNA accumulation or stability, evident in the weak accumulation of this phasi RNA position. Additional influences on this first phasi RNA duplex include the sequence composition and length, and we show that these factors impact Argonaute loading.

Plants possess different classes of endogenous small interfering RNAs (siRNAs) that play roles in regulating gene expression through gene silencing. Of these, a class of phased secondary siRNAs (phasiRNAs) are of particular interest because they are preferentially and highly abundant in the reproductive tissues of grass families. Two classes of reproductive phasiRNAs have been reported so far; the 21-nt accumulate prior to meiosis and are triggered by miR2118 while the 24-nt accumulate during meiosis and are triggered by miR2275. Even though phasiRNAs have been discovered about a decade ago, their functions and mechanism of action are yet to be identified. One of the challenges in studying these phasiRNAs is that; unlike microRNA precursors that produce a single microRNA duplex, phasiRNA precursors (PHAS) produce a number of phasiRNA duplexes, and for a plant like rice, there are thousands of these PHAS loci across the genome, necessitating the initial use of computational approaches for efficient experimental design. Also, the fact that PHAS loci originate from non-coding region of the genome, simple sequence comparison approaches are not effective. I employ an integrated approach to characterize phasiRNAs and their corresponding PHAS loci; 1) I use a comparative genomics approach to systematically compare rice and maize PHAS loci together with their phasiRNA products, 2) I use a synteny approach to extend the rice and maize comparison to perform a comprehensive PHAS loci cross-species comparison across ten diverse grass species, 3) I use a network approach to get a “holistic” view of the phasiRNA pathway and in turn identify key interacting partners and their interactions. With these approaches I was able to; demonstrate a number of phasiRNA characteristics, including their cis regulatory activity, establish PHAS loci relationships across diverse grass species, identify novel co-regulatory modules involving microRNAs, Transcription Factors (TFs), and PHAS genes. Such findings are important to further our understanding of plant reproductive biology and have potential to be applicable in biotech companies to produce hybrid crops with higher yields.

Although plants are able to withstand a range of environmental conditions, spikes in ambient temperature can impact plant fertility causing reductions in seed yield and notable economic losses 1 , 2 . Therefore, understanding the precise molecular mechanisms that underpin plant fertility under environmental constraints is critical to safeguarding future food production 3 . Here, we identified two Argonaute-like proteins whose activities are required to sustain male fertility in maize plants under high temperatures. We found that MALE-ASSOCIATED ARGONAUTE-1 and -2 associate with temperature-induced phased secondary small RNAs in pre-meiotic anthers and are essential to controlling the activity of retrotransposons in male meiocyte initials. Biochemical and structural analyses revealed how male-associated Argonaute activity and its interaction with retrotransposon RNA targets is modulated through the dynamic phosphorylation of a set of highly conserved, surface-located serine residues. Our results demonstrate that an Argonaute-dependent, RNA-guided surveillance mechanism is critical in plants to sustain male fertility under environmentally constrained conditions, by controlling the mutagenic activity of transposons in male germ cells. MALE-ASSOCIATED ARGONAUTE-1 and -2 promote heat-induced phasiRNA production, which represses heat-activated retrotransposons and protects male fertility. This activity is regulated by heat-mediated hypophosphorylation of the Argonaute proteins.

ABSTRACT Plant small RNAs are a diverse and complex set of molecules, ranging in length from 21 to 24 nt, involved in a wide range of essential biological processes. High-throughput sequencing is used for the discovery and quantification of small RNAs. However, several biases can occur during the preparation of small RNA libraries, especially using low input RNA. We used two stages of maize anthers to evaluate the performance of seven commercially-available methods for small RNA library construction, using different RNA input amounts. We show that when working with plant material, library construction methods have differing capabilities to capture small RNAs, and that different library construction methods provide better results when applied to the detection of microRNAs, phasiRNAs, or tRNA-derived fragment. We also observed that ligation bias occurs at both ends of miRNAs and phasiRNAs, suggesting that the biased compositions observed in small RNA populations, including nonstoichiometric levels of phasiRNAs within a locus, may reflect a combination of biological and technical influences. Competing Interest Statement The authors have declared no competing interest.

Environment-conditioned genic male sterility is a key strategy used to produce hybrid seeds efficiently in many crops, with the exception of maize. The underlying molecular mechanisms of environment-conditioned sterility are poorly understood. Here, we report a derivative line of the male sterile outer cell layer 4 (ocl4) mutant of maize, in which fertility was restored and perpetuated over several generations, under warm growing conditions. Conditionally fertile ocl4 anthers exhibit the anatomical abnormality of a partially duplicated endothecial layer, just like their sterile counterparts. We profiled the dynamics of phased, small interfering RNAs (phasiRNAs) during pre-meiotic development in fully sterile and various grades of semi-fertile ocl4 anthers. We found that the biogenesis of 21-nt phasiRNAs is largely dependent on Ocl4 at three key steps: (1) production of PHAS precursor transcripts, (2) expression of miR2118 that modulates precursor processing, and (3) accumulation of 21-nt phasiRNAs. We propose that 21-nt reproductive phasiRNAs buffer development under unfavorable environmental conditions and are dispensable under favorable conditions. Competing Interest Statement The authors have declared no competing interest.

The Solanaceae or nightshade family is an economically important group that harbors a remarkable amount of diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the family's small RNA genomic landscape, we downloaded over 255 publicly available small RNA datasets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major small RNA classes: (1) microRNAs (miRNAs), typically 20-22 nt RNAs generated from a hairpin precursor and functioning in gene silencing, and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs (hc-siRNAs) typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs (phasiRNAs) and trans-acting siRNAs (tasiRNAs) generated via miRNA-directed cleavage of a Pol II-derived RNA precursor. Our analyses described thousands of small RNA loci, including poorly-understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these small RNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar small RNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities. Competing Interest Statement The authors have declared no competing interest. Footnotes * This is a revised manuscript in response to reviewer comments.