Chiara Naro

The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.

The interplay between DNA damage response and RNA processing: the unexpected role of splicing factors as gatekeepers of genome stability.

Genome integrity is constantly threatened by endogenous and exogenous factors. However, its preservation is ensured by a network of pathways that prevent and/or repair the lesion, which constitute the DNA damage response (DDR). Expression of the key proteins involved in the DDR is controlled by numerous post-transcriptional mechanisms, among which pre-mRNA splicing stands out. Intriguingly, several splicing factors (SFs) have been recently shown to play direct functions in DNA damage prevention and repair, which go beyond their expected splicing activity. At the same time, evidence is emerging that DNA repair proteins (DRPs) can actively sustain the DDR by acting as post-transcriptional regulator of gene expression, in addition to their well-known role in the mechanisms of signaling and repair of the lesion. Herein, we will review these non-canonical functions of both SFs and DRPs, which suggest the existence of a tight interplay between splicing regulation and canonical DNA safeguard mechanisms ensuring genome stability.

EMT and stemness: flexible processes tuned by alternative splicing in development and cancer progression

Epithelial-to-mesenchymal transition (EMT) is associated with metastasis formation as well as with generation and maintenance of cancer stem cells. In this way, EMT contributes to tumor invasion, heterogeneity and chemoresistance. Morphological and functional changes involved in these processes require robust reprogramming of gene expression, which is only partially accomplished at the transcriptional level. Alternative splicing is another essential layer of gene expression regulation that expands the cell proteome. This step in post-transcriptional regulation of gene expression tightly controls cell identity between epithelial and mesenchymal states and during stem cell differentiation. Importantly, dysregulation of splicing factor function and cancer-specific splicing isoform expression frequently occurs in human tumors, suggesting the importance of alternative splicing regulation for cancer biology.In this review, we briefly discuss the role of EMT programs in development, stem cell differentiation and cancer progression. Next, we focus on selected examples of key factors involved in EMT and stem cell differentiation that are regulated post-transcriptionally through alternative splicing mechanisms. Lastly, we describe relevant oncogenic splice-variants that directly orchestrate cancer stem cell biology and tumor EMT, which may be envisioned as novel targets for therapeutic intervention.

An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation

Summary Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis.

Molecular mechanisms involved in HIV-1-Tat mediated inhibition of telomerase activity in human CD4+ T lymphocytes

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by a progressive decline of CD4(+) T cells and by other immune disorders that are similar to those observed during aging and which lead eventually to AIDS. One of the mechanisms involved in HIV-1 induced immunodeficiency may be the lack of telomerase induction and the consequent impairment of the potential required for CD4(+) T cell expansion. Telomerase compensates for the progressive telomere loss during cell division and preserves the replicative potential of T lymphocytes after repeated antigenic stimulation. The enzyme is activated by post-translational modifications, such as phosphorylation and also by the nuclear import of its catalytic subunit hTERT from the cytoplasm. In previous studies we found a reduction of telomerase activity in the nucleus of CD4(+) T cells infected with HIV-1 or non-infected but exposed to Tat protein. However, the mechanism for this loss of activity has not been elucidated yet. In the present study, we found that HIV-1 Tat inhibited telomerase activity in CD4(+) T cells by different mechanisms. First, it reduced nuclear levels of hTERT. Secondly, this protein perturbed the AKT pathway and the molecular interaction with the chaperones required for hTERT phosphorylation, nuclear import and activation. These results suggest that in addition to inducing direct cell death, HIV infection may also reduce the replicative potential of non-infected CD4(+) T cells and this may contribute to the overall immunodeficiency in AIDS patients.

Splicing Regulation: A Molecular Device to Enhance Cancer Cell Adaptation

Alternative splicing (AS) represents a major resource for eukaryotic cells to expand the coding potential of their genomes and to finely regulate gene expression in response to both intra- and extracellular cues. Cancer cells exploit the flexible nature of the mechanisms controlling AS in order to increase the functional diversity of their proteome. By altering the balance of splice isoforms encoded by human genes or by promoting the expression of aberrant oncogenic splice variants, cancer cells enhance their ability to adapt to the adverse growth conditions of the tumoral microenvironment. Herein, we will review the most relevant cancer-related splicing events and the underlying regulatory mechanisms allowing tumour cells to rapidly adapt to the harsh conditions they may face during the occurrence and development of cancer.

Dissecting a Hub for Immune Response: Modeling the Structure of MyD88

Immune cells sense foreign organisms through the evolutionarily conserved family of Toll-like receptors. Signaling from these receptors relies on oligomerization of adaptor molecules. In this issue of Structure, Vynke et al. (2016) shed light on the dynamical structure of the homo- and hetero-dimerization domain of MyD88, the main adaptor utilized by Toll-like receptors.

Timely-regulated intron retention as device to fine-tune protein expression

A key step in pre-mRNA processing is represented by splicing, the multilayered process operated by the spliceosome that removes the intervening non-coding introns and ligates adjacent exons. Splicing is necessary to yield a mature, translatable mRNA and its dysregulation underlies many human pathologies. Notably, weak conservation of the sequences defining the exon-intron boundaries allows flexibility in the recognition of many exons by the spliceosome. As a consequence, alternative splicing (AS) of such variable exons generates multiple mRNAs, with potentially different coding properties and patterns of expression, from most mammalian genes. Retention of select introns into mature mRNAs represents a peculiar pattern of AS that is emerging as a regulatory mechanism for developmentally-modulated gene expression patterns. Granulocyte differentiation provided one of the first examples of intron retention (IR) program set in motion to regulate gene expression. Transcripts encoding for proteins no longer required for granulopoiesis, and potentially interfering with it, are eliminated by the nonsense-mediated (NMD) pathway through IRmediated introduction of premature termination codons (PTCs). Similar coordinated and widespread dampening of specific set of genes through IR has been described for several differentiation programs or cellular responses to external stimuli. Spermatogenesis, however, represents a remarkable exception. Spermatogenesis involves profound genetic and morphological changes that are necessary for the differentiation of the male germ cell into a motile, fertile spermatozoon. Although proper progression of spermatogenesis requires the timely regulated expression of specific factors for each phase, transcription is not always active during this process. Indeed, nuclear condensation in post-meiotic male germ cells leads to a progressive decline of their transcriptional activity, which ultimately halts in spermatozoa. We have recently shown that an orchestrated IR program activated during meiosis contributes to temporally regulate the expression of genes during spermatogenesis. IR generates stable transcripts which persist in the nucleus of meiotic spermatocytes for several days after their synthesis, whose splicing and translation is delayed until the post-meiotic phases of spermatogenesis. In this way, meiotic IR acts as a compensatory mechanism for the transcriptional inactivity of the terminal phases of germ cell differentiation. Of note, IR-regulated genes encode for proteins that are crucial for proper development and functionality of the spermatozoon, such as those involved in the maturation of the flagellum or in sperm-egg recognition. Interestingly, robust accumulation in the nucleus of stable intron-retaining transcripts was also observed during the cellular response to heat shock. This observation suggests that IR stabilizes precursor transcripts before the global inhibition of RNA transcription caused by heat, and that their delayed splicing may promote efficient recovery of gene expression at the end of the stress. Furthermore, a “positive” role for IR was described in neurons. Posttranscriptional splicing of intron-retaining transcripts during neuronal activation allowed rapid expression of proteins encoded by genes that are too long to be rapidly transcribed, processed and translated in response to transient external stimuli. Thus, regulation of IR is emerging as a mechanism that can compensate both deficiencies and inefficiencies of the transcriptional process in eukaryotic cells. Notably, common traits of spermatogenic and neuronal IR programs are the nuclear preservation of intron-retaining transcripts and their protection from nuclear mechanisms of RNA surveillance. Therefore, it might be of interest to understand whether common mechanisms underlying these features exist in germ cells and neurons, possibly relying on the activity of splicing factors that are selectively expressed in these cells, such as PTBP2 or the STAR protein SLM2. Intron-retaining genes are expressed at higher levels than properly spliced genes in meiotic cells, and splicing of their weak introns is improved by reducing the transcriptional load through inhibition of the RNA polymerase II activity (Fig. 1). This finding suggests that an RNA synthetic activity exceeding the splicing capability of the cell represents the driver of the male meiotic IR program. Higher expression levels were also observed for heat shock-regulated intron-retaining genes and neuronal post-transcriptionally spliced pre-mRNAs. Thus, competition of introns for limiting splicing factors could represent a conserved mechanism controlling eukaryotic gene expression

Binding site density enables paralog-specific activity of SLM2 and Sam68 proteins in Neurexin2 AS4 splicing control

Abstract SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein–RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites.

Global changes in transcriptome regulation in postmeiotic spermatids

The male germ cell undergoes a proliferative step of amplification of the cell number, a meiotic step that reduces the genome to haploid, and a morphological differentiation stage to form spermatozoa. Although each phase requires a dynamic repertoire of functional factors, transcription is not always active in spermatogenesis, being characterized by a mitotic phase in spermatogonia, a meiotic phase in pachytene spermatocytes and a post-meiotic phase in round spermatids. We have performed deep-sequencing analysis of the transcriptome of purified populations of pachytene spermatocytes and round spermatids. We generated strend-specific RNAseq data for polyadenylated RNAs, resulting in >100 million 100bp mapped reads for each sample. Comparison of the spermatid versus spermatocytes transcriptome revealed 5508 up regulated and 7218 down-regulated genes in post-meiotic germ cells. Cell cycle and splicing were the most significantly up regulated pathways in post-meiotic versus meiotic cells. As for the splicing, 3278 exons in 2185 genes were differentially regulated in spermatids and spermatocytes. The most represented classes were alternative first exons, intron retention, alternative last exons and exon cassettes. These changes were accompanied by changes in the expression of key splicing factors, like the overall down regulation of the serine-arginine rich (SR) family, involved in positive regulation of constitutive and alternative splicing events. Our results indicate that male germ cells extensively modify the pattern of gene expression during the meiotic divisions and identify key molecular players involved in this transition. The global reduction in the SR protein family, known positive regulators of splicing events, may drive the extensive changes in alternative splicing observed between meiotic and post-meiotic germ cells.