Malat1 is a long-noncoding RNA with critical roles in gene regulation and cancer metastasis, however its functional role in stem cells is largely unexplored. We here perform a nuclear knockdown of Malat1 in mouse embryonic stem cells, causing the de-regulation of 320 genes and aberrant splicing of 90 transcripts, some of which potentially affecting the translated protein sequence. We find evidence that Malat1 directly interacts with gene bodies and aberrantly spliced transcripts, and that it locates upstream of down-regulated genes at their putative enhancer regions, in agreement with functional genomics data. Consistent with this, we find these genes affected at both exon and intron levels, suggesting that they are transcriptionally regulated by Malat1. Besides, the down-regulated genes are regulated by specific transcription factors and bear both activating and repressive chromatin marks, suggesting that some of them might be regulated by bivalent promoters. We propose a model in which Malat1 facilitates the transcription of genes involved in chromatid dynamics and mitosis in one pathway, and affects the splicing of transcripts that are themselves involved in RNA processing in a distinct pathway. Lastly, we compare our findings with Malat1 perturbation studies performed in other cell systems and in vivo.

MicroRNAs are ~22-nucleotide RNAs that bind to mRNAs and repress their expression. They are present in all multicellular animals studied in numbers that roughly scale with organismal complexity. Since microRNAs are rarely lost or changed in sequence during evolution, it is possible to study their features across vast evolutionary distances. We here leverage the curated MirGeneDB 3.0 database to analyze ~21,000 microRNAs from 114 species - ranging from sea anemone to human - spanning 800 million years of evolution. We study features related to their biogenesis, expression and function and present these using graphic Banner plots - validating previous studies and making new observations. We find strong correlations between microRNA evolutionary age, number of paralogs and expression, with old microRNAs such as Let-7 and Mir-10 being consistently more abundant than young ones. Mature microRNAs longer than 22 nucleotides tend to have more bulges, indicating that biogenesis recognizes their compressed tertiary structure. While younger microRNAs have seed sequences that resemble background genomic sequence, older microRNAs have a significant avoidance of GC-rich seeds that would confer overly strong target binding. Overall, we find that features for a given microRNA family tend to be constant across evolution, and that while younger microRNAs are individually more variable in their features, the evolutionarily old microRNAs appear to have been selected from a narrower spectrum of features.

Single-cell transcriptomics are increasingly being used to infer gene regulatory relationships - as regulators and their targets are expected to covary stochastically across cells. It has been assumed that a major confounder is the time lag between the measurements of the regulator at the RNA level to the production of the translated and active protein. We have recently conducted two studies in which we circumvent this time lag - by either measuring a regulator that is active as an RNA molecule or by profiling transcription factors at the protein level in single cells. Even as we circumvent the confounding time lag, we still find that our inferences are imperfect, suggesting that other substantial confounders remain. We discuss possible caveats and pitfalls in predicting gene regulation from single-cell transcriptomics data, and we suggest paths to more stringent benchmarking against biochemical and genetic perturbation data, and to using easily understood performance metrics such as accuracy, sensitivity and specificity. We foresee that greater stringency and standardization in benchmarking will drive the field forward.

The widespread protozoan Toxoplasma gondii chronically infects neural tissue in vertebrates and is linked to various neurological and neuropsychiatric disorders in humans. However, its effects on sparsely infected neurons and on broader neural circuits remain elusive. Our study reveals that T. gondii infection disrupts cytoskeletal dynamics in SH-SY5Y neuronal cells and primary cortical neurons. Infected neuronal cells undergo significant cytomorphological changes, including retraction of dendritic extensions and alterations in microtubule and F-actin networks, across both parasite genotypes I and II. These cytoskeletal alterations were notably diminished in cells exposed to T. gondii mutants with impaired secretion via the MYR translocon, and were independent of intraneuronal parasite replication. Moreover, a bystander effect was observed, with supernatants from T. gondii-challenged cells inducing similar cytoskeletal changes in uninfected cells. Analyses of extracellular vesicles (EVs) in supernatants revealed differential expression of host microRNAs in response to infection, most notably the upregulation of miR-221-3p, a microRNA not previously associated with T. gondii. The data indicate that unidentified parasite-derived effector(s) secreted via the MYR translocon, in conjunction with MYR-independently induced EV-associated host microRNAs, mediate cytoskeletal alterations in both infected and bystander neuronal cells. The findings provide new insights into molecular mechanisms by which T. gondii infection may disrupt neural networks, shedding light on its potential role in neuronal dysregulation.