Paola Zuccotti

The role of miR-103 and miR-107 in regulation of CDK5R1 expression and in cellular migration.

CDK5R1 encodes p35, a specific activator of the serine/threonine kinase CDK5, which plays crucial roles in CNS development and maintenance. CDK5 activity strongly depends on p35 levels and p35/CDK5 misregulation is deleterious for correct CNS function, suggesting that a tightly controlled regulation of CDK5R1 expression is needed for proper CDK5 activity. Accordingly, CDK5R1 expression was demonstrated to be controlled at both transcriptional and post-transcriptional levels, but a possible regulation through microRNAs (miRNAs) has never been investigated. We predicted, within the large CDK5R1 3′UTR several miRNA target sites. Among them, we selected for functional studies miR-103 and miR-107, whose expression has shown a strong inverse correlation with p35 levels in different cell lines. A significant reduction of CDK5R1 mRNA and p35 levels was observed after transfection of SK-N-BE neuroblastoma cells with the miR-103 or miR-107 precursor (pre-miR-103 or pre-miR-107). Conversely, p35 levels significantly increased following transfection of the corresponding antagonists (anti-miR-103 or anti-miR-107). Moreover, the level of CDK5R1 transcript shifts from the polysomal to the subpolysomal mRNA fraction after transfection with pre-miR-107 and, conversely, from the subpolysomal to the polysolmal mRNA fraction after transfection with anti-miR-107, suggesting a direct action on translation efficiency. We demonstrate, by means of luciferase assays, that miR-103 and miR-107 are able to directly interact with the CDK5R1 3′-UTR, in correspondence of a specific target site. Finally, miR-103 and miR-107 overexpression, as well as CDK5R1 silencing, caused a reduction in SK-N-BE migration ability, indicating that these miRNAs affect neuronal migration by modulating CDK5R1 expression. These findings indicate that miR-103 and miR-107 regulate CDK5R1 expression, allowing us to hypothesize that a miRNA-mediated mechanism may influence CDK5 activity and the associated molecular pathways.

Hyper conserved elements in vertebrate mRNA 3′-UTRs reveal a translational network of RNA-binding proteins controlled by HuR

Little is known regarding the post-transcriptional networks that control gene expression in eukaryotes. Additionally, we still need to understand how these networks evolve, and the relative role played in them by their sequence-dependent regulatory factors, non-coding RNAs (ncRNAs) and RNA-binding proteins (RBPs). Here, we used an approach that relied on both phylogenetic sequence sharing and conservation in the whole mapped 3′-untranslated regions (3′-UTRs) of vertebrate species to gain knowledge on core post-transcriptional networks. The identified human hyper conserved elements (HCEs) were predicted to be preferred binding sites for RBPs and not for ncRNAs, namely microRNAs and long ncRNAs. We found that the HCE map identified a well-known network that post-transcriptionally regulates histone mRNAs. We were then able to discover and experimentally confirm a translational network composed of RNA Recognition Motif (RRM)-type RBP mRNAs that are positively controlled by HuR, another RRM-type RBP. HuR shows a preference for these RBP mRNAs bound in stem–loop motifs, confirming its role as a ‘regulator of regulators’. Analysis of the transcriptome-wide HCE distribution revealed a profile of prevalently small clusters separated by unconserved intercluster RNA stretches, which predicts the formation of discrete small ribonucleoprotein complexes in the 3′-UTRs.

In Vivo Translatome Profiling in Spinal Muscular Atrophy Reveals a Role for SMN Protein in Ribosome Biology

Summary Genetic alterations impacting ubiquitously expressed proteins involved in RNA metabolism often result in neurodegenerative conditions, with increasing evidence suggesting that translation defects can contribute to disease. Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, whose role in pathogenesis remains unclear. Here, we identified in vivo and in vitro translation defects that are cell autonomous and SMN dependent. By determining in parallel the in vivo transcriptome and translatome in SMA mice, we observed a robust decrease in translation efficiency arising during early stages of disease. We provide a catalogue of RNAs with altered translation efficiency, identifying ribosome biology and translation as central processes affected by SMN depletion. This was further supported by a decrease in the number of ribosomes in SMA motor neurons in vivo. Overall, our findings suggest ribosome biology as an important, yet largely overlooked, factor in motor neuron degeneration.

Centaurin-α2 Interacts with β-Tubulin and Stabilizes Microtubules

Centaurin-α2 is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α2 can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α2 interacting protein, β-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of β-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α2 overexpression in HeLa cells and extraction of soluble (αβ dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α2 mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α2 remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α2 and tubulin confirmed that Centaurin-α2 promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α2 overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α2 role on MT stabilization. Centaurin-α2 interacts with β-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α2 as a new microtubule-associated protein (MAP) increasing MT stability.

Translational compensation of genomic instability in neuroblastoma

Cancer-associated gene expression imbalances are conventionally studied at the genomic, epigenomic and transcriptomic levels. Given the relevance of translational control in determining cell phenotypes, we evaluated the translatome, i.e., the transcriptome engaged in translation, as a descriptor of the effects of genetic instability in cancer. We performed this evaluation in high-risk neuroblastomas, which are characterized by a low frequency of point mutations or known cancer-driving genes and by the presence of several segmental chromosomal aberrations that produce gene-copy imbalances that guide aggressiveness. We thus integrated genome, transcriptome, translatome and miRome profiles in a representative panel of high-risk neuroblastoma cell lines. We identified a number of genes whose genomic imbalance was corrected by compensatory adaptations in translational efficiency. The transcriptomic level of these genes was predictive of poor prognosis in more than half of cases, and the genomic imbalances found in their loci were shared by 27 other tumor types. This homeostatic process is also not limited to copy number-altered genes, as we showed the translational stoichiometric rebalance of histone genes. We suggest that the translational buffering of fluctuations in these dose-sensitive transcripts is a potential driving process of neuroblastoma evolution.

Studying the Translatome with Polysome Profiling.

Polysome fractionation by sucrose density gradient centrifugation followed by analysis of RNA and protein is a technique that allows to understand the changes in translation of individual mRNAs as well as genome-wide effects on the translatome. Here, we describe the polysome profiling technique and RNA as well as protein isolation procedures from sucrose fractions.

HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA

Summary The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3′ UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.

The architecture of the human RNA-binding protein regulatory network

RNA-binding proteins (RBPs) are key players of post-transcriptional regulation of gene expression. These proteins influence both cellular physiology and pathology by regulating processes ranging from splicing and polyadenylation to mRNA localization, stability, and translation. To fine-tune the outcome of their regulatory action, RBPs rely on an intricate web of competitive and cooperative interactions. Several studies have described individual interactions of RBPs with RBP mRNAs, suggestive of a RBP-RBP regulatory structure. Here we present the first systematic investigation of this structure, based on a network including almost fifty thousand experimentally determined interactions between RBPs and bound RBP mRNAs. Our analysis identified two features defining the structure of the RBP-RBP regulatory network. What we call “RBP clusters” are groups of densely interconnected RBPs which co-regulate their targets, suggesting a tight control of cooperative and competitive behaviors. “RBP chains”, instead, are hierarchical structures driven by evolutionarily ancient RBPs, which connect the RBP clusters and could in this way provide the flexibility to coordinate the tuning of a broad set of biological processes. The combination of these two features suggests that RBP chains may use the modulation of their RBP targets to coordinately control the different cell programs controlled by the RBP clusters. Under this island-hopping model, the regulatory signal flowing through the chains hops from one RBP cluster to another, implementing elaborate regulatory plans to impact cellular phenotypes. This work thus establishes RBP-RBP interactions as a backbone driving post-transcriptional regulation of gene expression to allow the fine-grained control of RBPs and their targets.

An homeotic post-transcriptional network controlled by the RNA-binding protein RBMX

Post-transcriptional regulation (PTR) of gene expression is a powerful determinant of protein levels and cellular phenotypes. The 5’ and 3’ untranslated regions of the mRNA (UTRs) mediate this role through sequence and secondary structure elements bound by RNA-binding proteins (RBPs) and noncoding RNAs. While functional regions in the 3’UTRs have been extensively studied, the 5’UTRs are still relatively uncharacterized. To fill this gap, here we used a computational approach based on phylogenetic conservation to identify hyper-conserved elements in human 5’UTRs (5’HCEs). Our assumption, supported by the recovery of functionally characterized elements, was that 5’HCEs would represent evolutionarily stable and hence important PTR sites. We identified over 5000 short, clustered 5’HCEs occurring in approximately 10% of human protein-coding genes. Among these, homeotic genes were highly enriched. Indeed, 52 of the 258 characterized homeotic genes contained at least one 5’HCE, including members of all four Hox clusters and several other families. Homeotic genes are essential transcriptional regulators. They drive body plan and neuromuscular development, and the role of PTR in their expression is mostly unknown. By integrating computational and experimental approaches we then identified the RBMX RNA-binding protein as the initiator of a post-transcriptional cascade regulating many such homeotic genes. RBMX is known to control its targets by modulating transcript abundance and alternative splicing. Adding to that, we observed translational control as a novel mode of regulation by this RBP. This work thus establishes RBMX as a versatile master controller of homeotic genes and of the developmental processes they drive.

HuD is a neural enhancer of global translation acting on mTORC1-responsive genes and sponged by the Y3 small non-coding RNA

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. UV-crosslinking and analysis of cDNA (CRAC) generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells. HuD target sites were identified in 1304 mRNAs, predominantly in the 3’UTR, with enrichment for genes involved in protein synthesis and axonogenesis. HuD bound many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlated with the translational efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target was the abundant, small non-coding RNA Y3, which represented 70% of HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its activity. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA. Graphical abstract