BRAF is a widely studied oncogene and its functions are well characterized in several cellular contests and diseases. However the regulation of the expression of BRAF is mostly unknown. With the aim to understand the post-transcriptional regulation of BRAF, we performed 3’RACE in A375 melanoma cells and we found 2 different 3’UTRs: the one commonly reported in many data bases (Reference) and a new one that is only predicted (X1). The two 3’UTRs are completely different in sequence and length (120nt vs 1350nt). Furthermore, they are transcribed from exon 18 or thanks to an alternative splicing event occurring between exon 18 and a newly discovered exon 19 (X1). By using Real Time PCR, we confirmed the expression of both transcripts in melanoma and non-melanoma cell lines. Moreover, using RNA-SEQ data available at TCGA, we showed the co-expression of Reference and X1 BRAF transcripts also in human biopsies. Due to our discovery that BRAF exists in at least 2 different transcript variants, we decided to investigate further the expression of all the BRAF isoforms reported in NCBI and Ensembl. To do so, we took advantage of the RNA-seq data of more than 4,800 patients belonging to 9 different cancer types. We show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1 and BRAF-X2) that differ in the last part of their open reading frames, as well as in the length (BRAF-ref: 76nt; BRAF-X1 and X2: up to 7kb) and in the sequence of their 3’UTRs. In melanoma cells, the X1 isoform is expressed at the highest level, while the most prevalent among the three isoforms varies from one cancer type to another. Moreover, the relative abundance among the three BRAF isoforms is maintained in melanoma cells with acquired resistance to BRAF and MEK inhibitors driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. Besides their 3’UTRs, also the very last part of the coding sequences differ among the three isoforms. By immunoprecipitation of BRAF in A375 cells and subsequently Mass-SPEC analysis, we revealed the existence of Reference and X1 proteins which are expressed at similar levels, while X2 is not detectable because quicky degraded by the proteasome. Furthermore functional studies show that the two proteins account together for BRAF activities both in vitro and in vivo. Given the differences in length and sequence between the reference and the X1 3’UTR, we hypothesized that the two isoforms undergo different regulation mediated by RNA-binding proteins or non-coding RNAs. We focused on post-transcriptional regulation by microRNAs. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the expression of target messenger RNAs (mRNAs) and for this reason play a key role in virtually all cellular processes. In spite of the availability of several prediction algorithms, the identification of specific miRNA-target interactions remains a challenge. In order to overcome this problem we developed an innovative method, called miR-CATCH v2.0, for the high-throughput identification of microRNAs that bind a target transcript. The protocol is based on the affinity purification of the target mRNA and bound miRNAs by using two different pools of 3’biotinylated anti-sense DNA probes (ODD and EVEN). We designed 12 probes (6 ODD probes and 6 EVEN probes) for the purification of X1-3’UTR-miRNAs complexes and we performed three separate and independent captures in A375 metastatic melanoma cells. MicroRNAs were identified through small RNA-sequencing and the top-scoring miRNAs that resulted consistently enriched in all the captures will be validated in vitro and in vivo experiments.
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|Titolo:||Analysis of the expression of all BRAF transcript variants and of their implication in post-transcriptional regulation mediated by miRNAs in melanoma|
|Appare nelle tipologie:||8.1 Tesi Dottorato|
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