MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development.

Correct orientation of the mitotic spindle determines the plane of cellular cleavage and is crucial for organ development. In the developing cerebral cortex, spindle orientation defects result in severe neurodevelopmental disorders, but the precise mechanisms that control this important event are not fully understood. Here, we use a combination of high-content screening and mouse genetics to identify the miR-34/449 family as key regulators of mitotic spindle orientation in the developing cerebral cortex. By screening through all cortically expressed miRNAs in HeLa cells, we show that several members of the miR-34/449 family control mitotic duration and spindle rotation. Analysis of miR-34/449 knockout (KO) mouse embryos demonstrates significant spindle misorientation phenotypes in cortical progenitors, resulting in an excess of radial glia cells at the expense of intermediate progenitors and a significant delay in neurogenesis. We identify the junction adhesion molecule-A (JAM-A) as a key target for miR-34/449 in the developing cortex that might be responsible for those defects. Our data indicate that miRNA-dependent regulation of mitotic spindle orientation is crucial for cell fate specification during mammalian neurogenesis.

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells.

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

Target RNAs Strike Back on MicroRNAs.

MicroRNAs are extensively studied regulatory non-coding small RNAs that silence animal genes throughout most biological processes, typically doing so by binding to partially complementary sequences within target RNAs. A plethora of studies has described detailed mechanisms for microRNA biogenesis and function, as well as their temporal and spatial regulation during development. By inducing translational repression and/or degradation of their target RNAs, microRNAs can contribute to achieve highly specific cell- or tissue-specific gene expression, while their aberrant expression can lead to disease. Yet an unresolved aspect of microRNA biology is how such small RNA molecules are themselves cleared from the cell, especially under circumstances where fast microRNA turnover or specific degradation of individual microRNAs is required. In recent years, it was unexpectedly found that binding of specific target RNAs to microRNAs with extensive complementarity can reverse the outcome, triggering degradation of the bound microRNAs. This emerging pathway, named TDMD for Target RNA-Directed MicroRNA Degradation, leads to microRNA 3'-end tailing by the addition of A/U non-templated nucleotides, trimming or shortening from the 3' end, and highly specific microRNA loss, providing a new layer of microRNA regulation. Originally described in flies and known to be triggered by viral RNAs, novel endogenous instances of TDMD have been uncovered and are now starting to be understood. Here, we review our current knowledge of this pathway and its potential role in the control and diversification of microRNA expression patterns.

Molecular control of animal cell cytokinesis.

Cytokinesis is the process by which mitotic cells physically split in two following chromosome segregation. Dividing animal cells first ingress a cytokinetic furrow and then separate the plasma membrane by abscission. The general cytological events and several conserved molecular factors involved in cytokinesis have been known for many years. However, recent progress in microscopy, chemical genetics, biochemical reconstitution and biophysical methodology has tremendously increased our understanding of the underlying molecular mechanisms. We discuss how recent insights have led to refined models of the distinct steps of animal cell cytokinesis, including anaphase spindle reorganization, division plane specification, actomyosin ring assembly and contraction, and abscission. We highlight how molecular signalling pathways coordinate the individual events to ensure faithful partitioning of the genome to emerging daughter cells.

RNA Polymerase II Elongation at the Crossroads of Transcription and Alternative Splicing.

The elongation phase of transcription lies at the core of several simultaneous and coupled events leading to alternative splicing regulation. Although underestimated in the past, it is at this phase of the transcription cycle where complexes affecting the transcription machinery itself, chromatin structure, posttranscriptional gene regulation and pre-mRNA processing converge to regulate each other or simply to consolidate higher-order complexes and functions. This paper focuses on the multiple processes that take place during transcription elongation which ultimately regulate the outcome of alternative splicing decisions.

Regulation of alternative splicing by a transcriptional enhancer through RNA pol II elongation.

Promoters and enhancers are cis-acting elements that control gene transcription via complex networks of protein-DNA and protein-protein interactions. Whereas promoters deal with putting in place the RNA polymerase, both enhancers and promoters can control transcriptional initiation and elongation. We have previously shown that promoter structure modulates alternative splicing, strengthening the concept of a physical and functional coupling between transcription and splicing. Here we report that the promoter effect is due to the control of RNA pol II elongation. We found that the simian virus 40 (SV40) transcriptional enhancer, inserted in fibronectin (FN) minigene constructs transfected into mammalian cells, controls alternative splicing by inhibiting inclusion of the FN extra domain I (EDI) exon into mature mRNA. Deletion analysis of enhancer subdomains and competitions in vivo with excess of specific enhancer DNA subfragments demonstrate that the "minimal" enhancer, consisting of two 72-bp repeats, is responsible for the splicing effect. The 72-bp repeat region has been reported to promote RNA pol II elongation. When transcription is driven by the alpha-globin promoter linked to the SV40 enhancer, basal EDI inclusion and activation by the SR (Ser-Arg-rich) protein SF2/ASF are much lower than with other promoters. Deletion of only one of the two 72-bp repeats not only provokes higher EDI inclusion levels but allows responsiveness to SF2/ASF. These effects are the consequence of a decrease in RNA pol II elongation evidenced both by an increase in the proportions of shorter proximal over full length transcripts and by higher pol II densities upstream of the alternative exon detected by chromatin immunoprecipitation.

Multiple links between transcription and splicing.

Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.

Control of alternative pre-mRNA splicing by RNA Pol II elongation: faster is not always better.

The realization that the mammalian proteomic complexity is achieved with a limited number of genes demands a better understanding of alternative splicing regulation. Promoter control of alternative splicing was originally described by our group in studies performed on the fibronectin gene. Recently, other labs extended our findings to the cystic fibrosis, CD44 and CGRP genes strongly supporting a coupling between transcription and pre-mRNA splicing. A possible mechanism that would fit in these results is that the promoter itself is responsible for recruiting splicing factors, such as SR proteins, to the site of transcription, possibly through transcription factors that bind the promoter or the transcriptional enhancers. An alternative model, discussed more extensively in this review, involves modulation of RNA pol II (pol II) elongation rate. The model is supported by findings that cis- and trans- acting factors that modulate pol II elongation on a particular template also provoke changes in the alternative splicing balance of the encoded mRNAs.