Inferring targeting modes of Argonaute-loaded tRNA fragments.

Transfer RNA fragments (tRFs) are an emerging class of small RNA molecules derived from mature or precursor tRNAs. They are found across a wide range of organisms and tissues, in small RNA fraction or loaded to Argonaute in numbers comparable to microRNAs. Their functions and mechanisms of action are largely unknown, and results obtained on individual tRFs are often hard to generalize. Here we predicted binding mechanisms and specific target interaction sites of 26 human Argonaute-loaded tRFs of different types using large-scale meta-analyses of available experimental data. Strikingly, our findings matched all interaction sites detected in a recent experimental screen, confirming the validity of our computational approach. Such sites are primarily located on the 5' end of tRFs and often involve additional binding along the tRF length, similar to microRNAs. Indicative of multiple layers of regulation, diverse regulatory non-coding RNAs comprised a third of the tRF targets, with the rest being protein-coding transcripts. In the latter, coding sequence and 3' UTRs were the likely primary target regions, although we observed interactions of tRFs with 5' UTRs. Another novel phenomenon we report, a large number of putative interactions between tRFs and introns, is compatible with the roles of Argonaute in the nucleus. Further, observed tRF-intron binding modes suggest a mechanism of interaction of tRFs with Argonaute-dependent introns, and we predict here >20 candidate introns of this type. Taken together, these results present tRFs as regulatory molecules with a rich functional spectrum.

Noncanonical Activity of Med4 as a Gatekeeper of Metastasis through Epigenetic Control of Integrin Signaling.

Breast cancer metastatic relapse after a latency period, known as metastatic dormancy. Through genetic screening in mice, we identified the mediator complex subunit 4 (Med4) as a novel tumor-cell intrinsic gatekeeper in metastatic reactivation. Med4 downregulation effectively awakened dormant breast cancer cells, prompting macroscopic metastatic outgrowth in the lungs. Med4 depletion results in profound changes in nuclear size and three-dimensional chromatin architecture from compacted to relaxed states in contrast to the canonical function of the Mediator complex. These changes rewire the expression of extracellular matrix proteins, integrins, and signaling components resulting in integrin-mediated mechano-transduction and activation of YAP and MRTF. The assembly of stress fibers pulls on the nuclear membrane and contributes to reinforcing the overall chromatin modifications by Med4 depletion. MED4 gene deletions were observed in patients with metastatic breast cancer, and reduced MED4 expression correlates with worse prognosis, highlighting its significance as a potential biomarker for recurrence.

LncRNA Malat1 suppresses pyroptosis and T cell-mediated killing of incipient metastatic cells.

The contribution of antitumor immunity to metastatic dormancy is poorly understood. Here we show that the long noncoding RNA Malat1 is required for tumor initiation and metastatic reactivation in mouse models of breast cancer and other tumor types. Malat1 localizes to nuclear speckles to couple transcription, splicing and mRNA maturation. In metastatic cells, Malat1 induces WNT ligands, autocrine loops to promote self-renewal and the expression of Serpin protease inhibitors. Through inhibition of caspase-1 and cathepsin G, SERPINB6B prevents gasdermin D-mediated induction of pyroptosis. In this way, SERPINB6B suppresses immunogenic cell death and confers evasion of T cell-mediated tumor lysis of incipient metastatic cells. On-target inhibition of Malat1 using therapeutic antisense nucleotides suppresses metastasis in a SERPINB6B-dependent manner. These results suggest that Malat1-induced expression of SERPINB6B can titrate pyroptosis and immune recognition at metastatic sites. Thus, Malat1 is at the nexus of tumor initiation, reactivation and immune evasion and represents a tractable and clinically relevant drug target.

Engineered hydrogel reveals contribution of matrix mechanics to esophageal adenocarcinoma and identifies matrix-activated therapeutic targets.

Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying protumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform for identifying potential therapeutic targets to disrupt the contribution of protumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC and inform the development of therapeutics that target ECM stiffness in EAC.

p53 Gain-of-Function Mutation Induces Metastasis via BRD4-Dependent CSF-1 Expression.

TP53 mutations are frequent in esophageal squamous cell carcinoma (ESCC) and other SCCs and are associated with a proclivity for metastasis. Here, we report that colony-stimulating factor-1 (CSF-1) expression is upregulated significantly in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. The p53-R172H-dependent CSF-1 signaling, through its cognate receptor CSF-1R, increases tumor cell invasion and lung metastasis, which in turn is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). In Trp53R172H tumor cells, p53 occupies the Csf-1 promoter. The Csf-1 locus is enriched with histone 3 lysine 27 acetylation (H3K27ac), which is likely permissive for fostering an interaction between bromodomain-containing domain 4 (BRD4) and p53-R172H to regulate Csf-1 transcription. Inhibition of BRD4 not only reduces tumor invasion and lung metastasis but also reduces circulating CSF-1 levels. Overall, our results establish a novel p53-R172H-dependent BRD4-CSF-1 axis that promotes ESCC lung metastasis and suggest avenues for therapeutic strategies for this difficult-to-treat disease. SIGNIFICANCE: The invasion-metastasis cascade is a recalcitrant barrier to effective cancer therapy. We establish that the p53-R172H-dependent BRD4-CSF-1 axis is a mediator of prometastatic properties, correlates with patient survival and tumor stages, and its inhibition significantly reduces tumor cell invasion and lung metastasis. This axis can be exploited for therapeutic advantage. This article is featured in Selected Articles from This Issue, p. 2489.

ARIADNA: machine learning method for ancient DNA variant discovery.

Ancient DNA (aDNA) studies often rely on standard methods of mutation calling, optimized for high-quality contemporary DNA but not for excessive contamination, time- or environment-related damage of aDNA. In the absence of validated datasets and despite showing extreme sensitivity to aDNA quality, these methods have been used in many published studies, sometimes with additions of arbitrary filters or modifications, designed to overcome aDNA degradation and contamination problems. The general lack of best practices for aDNA mutation calling may lead to inaccurate results. To address these problems, we present ARIADNA (ARtificial Intelligence for Ancient DNA), a novel approach based on machine learning techniques, using specific aDNA characteristics as features to yield improved mutation calls. In our comparisons of variant callers across several ancient genomes, ARIADNA consistently detected higher-quality genome variants with fast runtimes, while reducing the false positive rate compared with other approaches.

Evolutionary adaptation revealed by comparative genome analysis of woolly mammoths and elephants.

Comparative genomics studies typically limit their focus to single nucleotide variants (SNVs) and that was the case for previous comparisons of woolly mammoth genomes. We extended the analysis to systematically identify not only SNVs but also larger structural variants (SVs) and indels and found multiple mammoth-specific deletions and duplications affecting exons or even complete genes. The most prominent SV found was an amplification of RNase L (with different copy numbers in different mammoth genomes, up to 9-fold), involved in antiviral defense and inflammasome function. This amplification was accompanied by mutations affecting several domains of the protein including the active site and produced different sets of RNase L paralogs in four mammoth genomes likely contributing to adaptations to environmental threats. In addition to immunity and defense, we found many other unique genetic changes in woolly mammoths that suggest adaptations to life in harsh Arctic conditions, including variants involving lipid metabolism, circadian rhythms, and skeletal and body features. Together, these variants paint a complex picture of evolution of the mammoth species and may be relevant in the studies of their population history and extinction.

Dynamics of tRNA fragments and their targets in aging mammalian brain.

Background: The progress of next-generation sequencing technologies has unveiled various non-coding RNAs that have previously been considered products of random degradation and attracted only minimal interest. Among small RNA families, microRNA (miRNAs) have traditionally been considered key post-transcriptional regulators. However, recent studies have reported evidence for widespread presence of fragments of tRNA molecules (tRFs) across a range of organisms and tissues, and of tRF involvement in Argonaute complexes.  Methods:To elucidate potential tRF functionality, we compared available RNA sequencing datasets derived from the brains of young, mid-aged and old rats. Using sliding 7-mer windows along a tRF, we searched for putative seed sequences with high numbers of conserved complementary sites within 3' UTRs of 23 vertebrate genomes. We analyzed Gene Ontology term enrichment of predicted tRF targets and compared their transcript levels with targets of miRNAs in the context of age.  Results and Discussion: We detected tRFs originating from 3'- and 5'-ends of tRNAs in rat brains at significant levels. These fragments showed dynamic changes: 3' tRFs monotonously increased with age, while 5' tRFs displayed less consistent patterns. Furthermore, 3' tRFs showed a narrow size range compared to 5' tRFs, suggesting a difference in their biogenesis mechanisms. Similar to our earlier results in Drosophila and compatible with other experimental findings, we found "seed" sequence locations on both ends of different tRFs. Putative targets of these fragments were found to be enriched in neuronal and developmental functions. Comparison of tRFs and miRNAs increasing in abundance with age revealed small, but distinct changes in brain target transcript levels for these two types of small RNA, with the higher proportion of tRF targets decreasing with age. We also illustrated the utility of tRF analysis for annotating tRNA genes in sequenced genomes.

Age-driven modulation of tRNA-derived fragments in Drosophila and their potential targets.

BACKGROUND: Development of sequencing technologies and supporting computation enable discovery of small RNA molecules that previously escaped detection or were ignored due to low count numbers. While the focus in the analysis of small RNA libraries has been primarily on microRNAs (miRNAs), recent studies have reported findings of fragments of transfer RNAs (tRFs) across a range of organisms. RESULTS: Here we describe Drosophila melanogaster tRFs, which appear to have a number of structural and functional features similar to those of miRNAs but are less abundant. As is the case with miRNAs, (i) tRFs seem to have distinct isoforms preferentially originating from 5' or 3' end of a precursor molecule (in this case, tRNA), (ii) ends of tRFs appear to contain short "seed" sequences matching conserved regions across 12 Drosophila genomes, preferentially in 3' UTRs but also in introns and exons; (iii) tRFs display specific isoform loading into Ago1 and Ago2 and thus likely function in RISC complexes; (iii) levels of loading in Ago1 and Ago2 differ considerably; and (iv) both tRF expression and loading appear to be age-dependent, indicating potential regulatory changes from young to adult organisms. CONCLUSIONS: We found that Drosophila tRF reads mapped to both nuclear and mitochondrial tRNA genes for all 20 amino acids, while previous studies have usually reported fragments from only a few tRNAs. These tRFs show a number of similarities with miRNAs, including seed sequences. Based on complementarity with conserved Drosophila regions we identified such seed sequences and their possible targets with matches in the 3'UTR regions. Strikingly, the potential target genes of the most abundant tRFs show significant Gene Ontology enrichment in development and neuronal function. The latter suggests that involvement of tRFs in the RNA interfering pathway may play a role in brain activity or brain changes with age.