Sorting and packaging of RNA into extracellular vesicles shape intracellular transcript levels.

BACKGROUND: Extracellular vesicles (EVs) are released by nearly every cell type and have attracted much attention for their ability to transfer protein and diverse RNA species from donor to recipient cells. Much attention has been given so far to the features of EV short RNAs such as miRNAs. However, while the presence of mRNA and long noncoding RNA (lncRNA) transcripts in EVs has also been reported by multiple different groups, the properties and function of these longer transcripts have been less thoroughly explored than EV miRNA. Additionally, the impact of EV export on the transcriptome of exporting cells has remained almost completely unexamined. Here, we globally investigate mRNA and lncRNA transcripts in endothelial EVs in multiple different conditions. RESULTS: In basal conditions, long RNA transcripts enriched in EVs have longer than average half-lives and distinctive stability-related sequence and structure characteristics including shorter transcript length, higher exon density, and fewer 3' UTR A/U-rich elements. EV-enriched long RNA transcripts are also enriched in HNRNPA2B1 binding motifs and are impacted by HNRNPA2B1 depletion, implicating this RNA-binding protein in the sorting of long RNA to EVs. After signaling-dependent modification of the cellular transcriptome, we observed that, unexpectedly, the rate of EV enrichment relative to cells was altered for many mRNA and lncRNA transcripts. This change in EV enrichment was negatively correlated with intracellular abundance, with transcripts whose export to EVs increased showing decreased abundance in cells and vice versa. Correspondingly, after treatment with inhibitors of EV secretion, levels of mRNA and lncRNA transcripts that are normally highly exported to EVs increased in cells, indicating a measurable impact of EV export on the long RNA transcriptome of the exporting cells. Compounds with different mechanisms of inhibition of EV secretion affected the cellular transcriptome differently, suggesting the existence of multiple EV subtypes with different long RNA profiles. CONCLUSIONS: We present evidence for an impact of EV physiology on the characteristics of EV-producing cell transcriptomes. Our work suggests a new paradigm in which the sorting and packaging of transcripts into EVs participate, together with transcription and RNA decay, in controlling RNA homeostasis and shape the cellular long RNA abundance profile.

The thyroid gland and thyroid hormones in sheepshead minnow (Cyprinodon variegatus) during early development and metamorphosis.

The sheepshead minnow is widely used in ecotoxicological studies that only recently have begun to focus on disruption of the thyroid axis by xenobiotics and endocrine disrupting compounds. However, reference levels of the thyroid prohormone thyroxine (T4) and biologically active hormone 3,5,3'-triiodothyronine (T3) and their developmental patterns are unknown. This study set out to describe the ontogeny and morphology of the thyroid gland in sheepshead minnow, and to correlate these with whole-body concentrations of thyroid hormones during early development and metamorphosis. Eggs were collected by natural spawning in our laboratory. T4 and T3 were extracted from embryos, larvae and juveniles and an enzyme-linked immunoassay was used to measure whole-body hormone levels. Length and body mass, hatching success, gross morphology, thyroid hormone levels and histology were measured. The onset of metamorphosis at 12-day post-hatching coincided with surges in whole-body T4 and T3 concentrations. Thyroid follicles were first observed in pre-metamorphic larvae at hatching and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Follicle size and thyrocyte epithelial cell heights varied during development, indicating fluctuations in thyroid hormone synthesis activity. The increase in the whole-body T3/T4 ratio was indicative of an increase in outer ring deiodination activity. This study establishes a baseline for thyroid hormones in sheepshead minnows, which will be useful for the understanding of thyroid hormone functions and in future studies of thyroid toxicants in this species.

The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression.

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.