Cue-specific remodeling of the neuronal transcriptome through intron retention programs

Sub-cellular compartmentalization through the nuclear envelope has for a long time been primarily considered a physical barrier that separates nuclear and cytosolic contents. More recently, nuclear compartmentalization has emerged to harbor key regulatory functions in gene expression. A sizeable proportion of protein-coding mRNAs is more prevalent in the nucleus than in the cytosol reflecting the existence of mechanisms to control mRNA release into the cytosol. However, the biological relevance of the nuclear retention of mRNAs remains unclear. Here, we provide a comprehensive map of the subcellular localization of mRNAs in mature neurons and reveal that transcripts stably retaining introns are broadly targeted for nuclear retention. We systematically probed these transcripts upon neuronal stimulation and found that sub-populations of nuclear-retained transcripts are bi-directionally regulated in response to cues: some appear targeted for degradation while others undergo splicing completion to generate fully mature mRNAs which are exported to the cytosol to increase functional gene expression. Remarkably, different forms of stimulation mobilize distinct groups of intron-retaining transcripts and this selectivity arises from the activation of specific signaling pathways. Overall, our findings uncover cue-specific control of intron retention as a major regulator of acute remodeling of the neuronal transcriptome.

Nematode Small RNA Pathways in the Absence of piRNAs

Small RNA pathways play diverse regulatory roles in the nematode C. elegans. However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 5'-triphosphate small RNAs (22-24G-RNAs). These Ascaris WAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Ascaris CSR-1 likely functions to "license" gene expression in the absence of an Ascaris piRNA pathway. Ascaris ALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably, Ascaris WAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.

ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5' splice site motif within nuclear speckles

Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5' splice site (5'SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Here we demonstrate that these transcripts require the zinc finger protein ZFC3H1 for their decay and nuclear retention into nuclear speckles. Furthermore, we find that U1-70K, a component of the U1 snRNP spliceosomal complex, is also required for their nuclear retention and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of mRNAs with 5'SS motifs. Together, our results suggest a model where mRNAs with 5'SS motifs are recognized by U1 snRNP, which then acts with ZFC3H1 to both promote their decay and prevent nuclear export of these mRNAs by sequestering them in nuclear speckles. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs.

Platelets and extracellular vesicles and their cross-talk with cancer

Platelets play significant and varied roles in cancer progression, as detailed throughout this review series, via direct interactions with cancer cells as well as by long-range indirect interactions mediated by platelet releasates. Microvesicles (MV, also referred to as microparticles) released from activated platelets have emerged as major contributors to the platelet-cancer nexus. Interactions of platelet-derived MV (PMV) with cancer cells can promote disease progression through multiple mechanisms, but PMV also harbor anti-tumor functions. This complex relationship derives from the abilities of PMV both to bind to cancer cells as well as to non-transformed cells in the tumor microenvironment, and to transfer platelet-derived contents to the target cell, each of which can have stimulatory or modulatory effects. MV are extracellular vesicles of heterogeneous size, ranging from 100 nm to 1 µm in diameter, shed by living cells by outward budding of the plasma membrane, entrapping local cytosolic contents in an apparently stochastic manner. Hence, PMV are encapsulated by a lipid bilayer harboring surface proteins and lipids mirroring the platelet exterior, with internal components including platelet-derived mature mRNAs and pre-mRNAs, microRNAs (miRNAs) and other non-coding RNAs, proteins, second messengers, and mitochondria. Each of these elements engages in established and putative PMV functions in cancer. In addition, PMV contribute to cancer co-morbidities due to their roles in coagulation and thrombosis, and via interactions with inflammatory cells. However, separating effects of PMV from those of platelets in cancer contexts continues to be a major hurdle (Figure 1). This review will summarize our emerging understanding of the complex roles of PMV in the development and progression of cancer and cancer co-morbidities.

Nuclear retention of pre-mRNA involving Cajal bodies during meiotic prophase in plants

Gene regulation ensures that the appropriate genes are expressed at the proper times. Nuclear retention of incompletely spliced or mature mRNAs emerges as a novel, previously underappreciated layer of post-transcriptional gene regulation. Studies on this phenomenon indicated that it exerted significant impact on the regulation of gene expression by regulating export and translation delay, which allows synthesis of specific proteins in response to a stimulus, e.g. under stress conditions or at strictly controlled time points, e.g. during cell differentiation or development. Here, we found that transcription in microsporocytes, during prophase of the first meiotic division, occurs in pulsatile manner. After each pulse, the transcriptional activity is silenced, but the transcripts synthesized at this time are not exported immediately to the cytoplasm, but are retained in the nucleoplasm and Cajal bodies (CBs). In contrast to nucleoplasm, mature transcripts were not found in CBs. Only non-fully-spliced transcripts with retained introns were stored in the CBs. Retained introns are spliced at precisely defined times, and fully mature mRNAs are released into the cytoplasm, where the proteins are produced. These proteins are necessary for further cell development during meiotic prophase. Our findings provide new insight into the regulatory mechanisms of gene expression based on mRNA retention in the nucleus during the development of generative cells in plants. Similar processes were observed during spermatogenesis in animals. This indicates the existence of an evolutionarily conserved mechanism of gene expression regulation during generative cells development in Eukaryota.

Mitochondrial mRNA Processing in the Chlorophyte Alga Pediastrum duplex and Streptophyte Alga Chara vulgaris Reveals an Evolutionary Branch in Mitochondrial mRNA Processing

Mitochondria carry the remnant of an ancestral bacterial chromosome and express those genes with a system separate and distinct from the nucleus. Mitochondrial genes are transcribed as poly-cistronic primary transcripts which are post-transcriptionally processed to create individual translationally competent mRNAs. Algae post-transcriptional processing has only been explored in Chlamydomonas reinhardtii (Class: Chlorophyceae) and the mature mRNAs are different than higher plants, having no 5′ UnTranslated Regions (UTRs), much shorter and more variable 3′ UTRs and polycytidylated mature mRNAs. In this study, we analyzed transcript termini using circular RT-PCR and PacBio Iso-Seq to survey the 3′ and 5′ UTRs and termini for two green algae, Pediastrum duplex (Class: Chlorophyceae) and Chara vulgaris (Class: Charophyceae). This enabled the comparison of processing in the chlorophyte and charophyte clades of green algae to determine if the differences in mitochondrial mRNA processing pre-date the invasion of land by embryophytes. We report that the 5′ mRNA termini and non-template 3′ termini additions in P. duplex resemble those of C. reinhardtii, suggesting a conservation of mRNA processing among the chlorophyceae. We also report that C. vulgaris mRNA UTRs are much longer than chlorophytic examples, lack polycytidylation, and are polyadenylated similar to embryophytes. This demonstrates that some mitochondrial mRNA processing events diverged with the split between chlorophytic and streptophytic algae.

The secreted endoribonuclease ENDU-2 from the soma protects germline immortality in C. elegans

Multicellular organisms coordinate tissue specific responses to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, recent reports implicated release of small RNAs in regulating gene expression across tissue boundaries. Here, we show that the conserved poly-U specific endoribonuclease ENDU-2 in C. elegans is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 regulates gene expression across tissue boundaries in response to temperature alterations and contributes to maintenance of stem cell immortality, probably via retaining a stem cell specific program of gene expression.

The spliceosome inhibitors isoginkgetin and pladienolide B induce ATF3-dependent cell death

The spliceosome assembles on pre-mRNA in a stepwise manner through five successive pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the spliceosome have been identified and they are cytotoxic. However, little is known about genetic determinants of cell sensitivity. Activating transcription factor 3 (ATF3) is a transcription factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, we used a genetic approach to determine if ATF3 was important in determining the sensitivity of mouse embryonic fibroblasts (MEFs) to two splicing inhibitors: pladienolide B (PB) and isoginkgetin (IGG), that target different pre-spliceosome complexes. Both compounds led to increased ATF3 expression and apoptosis in control MEFs while ATF3 null cells were significantly protected from the cytotoxic effects of these drugs. Similarly, ATF3 was induced in response to IGG and PB in the two human tumour cell lines tested while knockdown of ATF3 protected cells from both drugs. Taken together, ATF3 appears to contribute to the cytotoxicity elicited by these spliceosome inhibitors in both murine and human cells.

A secreted endoribonuclease ENDU-2 from the soma protects germline immortality in C. elegans

Multicellular organisms coordinate tissue specific response to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, secreted small RNAs have recently been reported to regulate gene expression across tissue boundaries. Here we show that the conserved poly-U specific endoribonuclease ENDU-2 is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature in C. elegans. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 transmits environmental information across tissue boundaries and contributes to maintenance of stem cell immortality probably via retaining a stem cell specific program of gene expression.

Mutations primarily alter the inclusion of alternatively spliced exons

Genetic analyses and systematic mutagenesis have revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, consistent with the concept that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between genome sequence variation and exon inclusion across the transcriptome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across primary transcripts but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.