A widespread repression of miRNA has been described in almost every type of cancer, caused by promoter silencing, transcriptional regulation or alterations in miRNA processing. Our hypothesis is that also altered miRNAs degradation contributes to miRNA repression in cancer. This idea was suggested by recent data showing that miRNAs can be degraded upon interaction with their targets, if the targets fulfill precise requirements in terms of complementarity. We developed a way to circumvent this problem by exploiting 4S-Uridine (4SU), a low-frequency naturally occurring modified base that can be incorporated in place of Uridine into the nascent RNA. 4SU serves as an attachment point for a thiol-specific biotinylation that allow the separation of newly transcribed RNA from the total RNA population. This method was originally developed to study the dynamics of transcription and decay of mRNAs. We adapted the protocol to microRNAs: 4SU is added to the medium for a period of time sufficient to label also mature microRNAs (3hrs), then 4SU is removed from the medium and the decay of labeled species is monitored, at different time points, during the chase period. We can isolate enough materials to generate high-throughput sequencing libraries, thus we can simultaneously analyze the decay of all expressed miRNAs and their 3’ modified forms (“isomiRs”, that can display differential stability compared to canonical miRNAs). We applied this approach to analyze the dynamics of miRNA decay under physiological conditions (growing vs. quiescent cells) and under oncogenic stimuli.
In this project, I propose to rule out the hypothesis that endogenous RNA targets could regulate miRNAs by a target-induced miRNA decay (TIMD) mechanism. By a rigorous pipeline that combines a series of bioinformatics and experimental analyses, I will identify and validate a group of endogenous targets involved in TIMD.To explicitly validate this hypothesis, our team will exploit CRISPR/Cas9 gene editing to delete miRNA-responsive elements (MREs) from the 3'UTR of candidate transcripts, to specifically interfere with miRNA:target interaction while keeping mRNA and miRNA expression at their endogenous levels (WP1). State-of-the-art quantitation of gene and miRNA expression by digital PCR and RNA-sequencing will be used to measure miRNA and target expression upon manipulation. Additional tools specifically developed in our lab, namely IsomiRage (for the analysis of miRNA isoforms by small-RNA sequencing) and 4SU-labelling (to monitor miRNA decay), will be also exploited to achieve a comprehensive characterization of the bidirectional effects associated with target:miRNA interaction.
AWARDS
PATENTS
L’Istituto Italiano di Tecnologia (IIT) è una fondazione di diritto privato - cfr. determinazione Corte dei Conti 23/2015 “IIT è una fondazione da inquadrare fra gli organismi di diritto pubblico con la scelta di un modello di organizzazione di diritto privato per rispondere all’esigenza di assicurare procedure più snelle nella selezione non solo nell’ambito nazionale dei collaboratori, scienziati e ricercatori ”.
IIT è sotto la vigilanza del Ministero dell'Istruzione, dell'Università e della Ricerca e del Ministero dell'Economia e delle Finanze ed è stato istituito con la Legge 326/2003. La Fondazione ha l'obiettivo di promuovere l'eccellenza nella ricerca di base e in quella applicata e di favorire lo sviluppo del sistema economico nazionale. La costruzione dei laboratori iniziata nel 2006 si è conclusa nel 2009.
Lo staff complessivo di IIT conta circa 1440 persone. L’area scientifica è rappresentata da circa l’85% del personale. Il 45% dei ricercatori proviene dall’estero: di questi, il 29% è costituito da stranieri provenienti da oltre 50 Paesi e il 16% da italiani rientrati. Oggi il personale scientifico è composto da circa 60 principal investigators, circa 110 ricercatori e tecnologi di staff, circa 350 post doc, circa 500 studenti di dottorato e borsisti, circa 130 tecnici. Oltre 330 posti su 1400 creati su fondi esterni. Età media 34 anni. 41% donne / 59 % uomini.
Nel 2015 IIT ha ricevuto finanziamenti pubblici per circa 96 milioni di euro (80% del budget), conseguendo fondi esterni per 22 milioni di euro (20% budget) provenienti da 18 progetti europei, 17 finanziamenti da istituzioni nazionali e internazionali, circa 60 progetti industriali
La produzione di IIT ad oggi vanta circa 6990 pubblicazioni, oltre 130 finanziamenti Europei e 11 ERC, più di 350 domande di brevetto attive, oltre 12 start up costituite e altrettante in fase di lancio. Dal 2009 l’attività scientifica è stata ulteriormente rafforzata con la creazione di dieci centri di ricerca nel territorio nazionale (a Torino, Milano, Trento, Parma, Roma, Pisa, Napoli, Lecce, Ferrara) e internazionale (MIT ed Harvard negli USA) che, unitamente al Laboratorio Centrale di Genova, sviluppano i programmi di ricerca del piano scientifico 2015-2017.
Istituto Italiano di Tecnologia (IIT) is a public research institute that adopts the organizational model of a private law foundation. IIT is overseen by Ministero dell'Istruzione, dell'Università e della Ricerca and Ministero dell'Economia e delle Finanze (the Italian Ministries of Education, Economy and Finance). The Institute was set up according to Italian law 326/2003 with the objective of promoting excellence in basic and applied research andfostering Italy’s economic development. Construction of the Laboratories started in 2006 and finished in 2009.
IIT has an overall staff of about 1,440 people. The scientific staff covers about 85% of the total. Out of 45% of researchers coming from abroad 29% are foreigners coming from more than 50 countries and 16% are returned Italians. The scientific staff currently consists of approximately 60 Principal Investigators, 110 researchers and technologists, 350 post-docs and 500 PhD students and grant holders and 130 technicians. External funding has allowed the creation of more than 330 positions . The average age is 34 and the gender balance proportion is 41% female against 59% male.
In 2015 IIT received 96 million euros in public funding (accounting for 80% of its budget) and obtained 22 million euros in external funding (accounting for 20% of its budget). External funding comes from 18 European Projects, other 17 national and international competitive projects and approximately 60 industrial projects.
So far IIT accounts for: about 6990 publications, more than 130 European grants and 11 ERC grants, more than 350 patents or patent applications, 12 up start-ups and as many which are about to be launched. The Institute’s scientific activity has been further strengthened since 2009 with the establishment of 11 research nodes throughout Italy (Torino, Milano, Trento, Parma, Roma, Pisa, Napoli, Lecce, Ferrara) and abroad (MIT and Harvard University, USA), which, along with the Genoa-based Central Lab, implement the research programs included in the 2015-2017 Strategic Plan.
