Constitutive splicing is favored in strong splice sites, which have the conserved consensus sequences at 5 0 (CAG|GUAAGU) and 3 0 (NYAG|G) splice sites. Exons are usually shorter sequences, ranging from 50 to 250 bp, whereas introns can have broader length variation, from <100 bp to more than 1,000 bp, and around 20% of human introns are larger than 5 kb (Lander et al., 2001). In addition to constitutive exon splicing, the presence of alternative splice sites leads to variable ways to mature an mRNA ( Figure 2). Exons and introns might host alternative splice sites, which are similar to, but usually not as strong as, consensus splice sites (Fu & Ares, 2014;Kornblihtt et al., 2013). If a 5 0 or 3 0 alternative site lies within an exon, part of this exon might be read as an intron. Also, alternative splicing can lead to exon skipping, in which a constitutive 3 0 splice site fails to be recognized and assembly of spliceosome proceeds to the next available 3 0 splice site, which can result in exclusion of an exon. Alternatively, 5 0 splice sites might not be read as well, resulting in intron retention. In some transcripts, a mutual exclusion of exons might take place generating different messages . Cryptic splice sites are similar to consensus sites, but they are usually recognized only after a mutation in the gene, or of a spliceosome component, leading to alterations in the final message. In breast cancer, mutations in splicing factor 3B subunit 1 (SF3B1), for example, induce the use of cryptic 3 0 splice sites (DeBoever et al., 2015). Single nucleotide variants (SNVs) of 1,812 tumor samples were analyzed by whole-exome sequencing and revealed exon skipping and intron retention were among the most frequently observed alternative splicing events (Jung et al., 2015; Box 1).Alternative splicing mechanisms are dependent on recognition of splice site sequences in the pre-mRNA, which in turn depends on both the strength of sequences as well as the presence of proteins and signals that guide the spliceosome to these regions. Alternative exons in humans and mice are surrounded by more conserved regulatory sequences than constitutive exons, indicating the former ones are under strict regulation. Consistently, 30-50% of ISEs are located nearby alternative exons (Yeo, Van Nostrand, & Liang, 2007). Interestingly, ISEs and ESSs show a positional overlap in different pre-mRNA substrates, indicating the regulation depends on their positioning with respect to the 5 0 upstream exon and the bound proteins (Y. Wang et al., 2012). The ISSs can interfere with exon inclusion in different pre-mRNAs, and regulate 5 0 splice site choice, therefore, exerting alternative splicing regulation (Y. Wang et al., 2013). In this sense, mutations in exonic and intronic sequences are critical and might affect the balance of regulatory sequences and also their activity, since most effects in splicing are location-dependent. Intronic mutations are commonly associated with nonsense-mediated decay responses, caused by an increased rate of premature stop ...

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are essential players in the regulation of gene expression. The majority of the twenty different hnRNP proteins act through the modulation of pre‐mRNA splicing. Most have been shown to regulate the expression of critical genes for the progression of tumorigenic processes and were also observed to be overexpressed in several types of cancer. Moreover, these proteins were described as essential components for the maturation of some microRNAs (miRNAs). In the human genome, over 70% of miRNAs are transcribed from introns; therefore, we hypothesized that regulatory proteins involved with splicing could be important for their maturation. Increased expression of the miR‐17‐92 cluster has already been shown to be related to the development of many cancers, such as thyroid, lung, and lymphoma. In this article, we show that overexpression of hnRNP A1 and hnRNP C in BCPAP thyroid cancer cells directly affects the expression of miR‐17‐92 miRNAs. Both proteins associate with the 5′‐end of this cluster, strongly precipitate miRNAs miR‐17 and miR‐18a and upregulate the expression of miR‐92a. Upon overexpression of these hnRNPs, BCPAP cells also show increased proliferation, migration, and invasion rates, suggesting upregulation of these proteins and miRNAs is related to an enhanced tumorigenic phenotype.

BACKGROUND/AIMS: Pre-mRNA splicing is an essential step in eukaryotic gene expression regulation. Genes are composed of exons that remain in the mature mRNAs and intervening sequences named introns. Splicing is the removal of introns and ligation of exons in a mature transcript. Splice site or spliceosome component mutations can lead to different diseases, including neurodegenerative diseases and several cancer types. HuR is an RNA-binding protein that preferentially binds to U- and AU-rich elements, usually found at the 3' UTRs of some mRNAs. We previously observed HuR specifically associated with spliceosomes assembled on introns containing miR-18a and miR-19a. miR-18a and miR-19a are components of the intronic miR-17-92 cluster, along with other five miRNAs. This cluster has been reported to regulate proliferation, migration, and angiogenesis in cells. In this context, we reasoned HuR could be controlling the splicing and processing of these miRNAs, leading to altered cellular phenotypes. METHODS: We induced HuR overexpression in BCPAP and HEK-293T and analyzed the expression of miRNAs using qPCR, as well as the phenotypic effects in those cells. Cell counting to analyze cell growth was performed after trypan blue staining. Migration and invasion assays were performed using transwell filters and cells were counted after staining with crystal violet. We knocked down HuR using a specific siRNA and analyzed expression of miRNAs by qPCR, as well as cellular kinetics. RESULTS: Our results revealed HuR is associated with miR-19a in BCPAP and HEK-293T cells. Conversely, silencing HuR led to reduced miR-17-5p and miR-19a in BCPAP cells. Our data support that HuR stimulates the expression of miR-19, which is further processed and capable of finding its target sequence in a reporter plasmid. Cells overexpressing HuR showed increased cellular proliferation, migration, and invasion rates. Notably, under the presence of antimiR-19a, BCPAP-HuR cells showed reduced cell growth. Taken together, these results indicate the molecular alterations observed are associated with upregulation of miR-19a, leading to cellular processes involved in cancer development. CONCLUSION: Our findings propose a connection between HuR, miRNA biogenesis and cellular modifications. HuR stimulates miR-19a and miR-19b expression, which leads to up-regulation of cell proliferation, migration and invasion, promoting cancer development.

O splicing é um processo crítico para expressão gênica em eucariotos, pois os genes são transcritos como pré-mRNAs, os quais são compostos por exons (sequências que permanecem nos mRNAs maduros) e introns (sequências intermediárias). O processo de splicing envolve a excisão de introns e a ligação de exons, resultando em transcritos maduros (mRNA). Este processo é realizado por uma maquinaria complexa chamada spliceossomo, que é composta por cinco pequenos RNAs associados a proteínas (snRNAs) e mais de 100 proteínas, sendo que algumas podem associar-se transitoriamente a componentes de spliceossomo ou aos pré-mRNAs. As hnRNPs são proteínas que podem associar-se ao complexo spliceossomo, desempenhando um papel regulador nos locais de splicing e frequentemente envolvidas na mediação de splicing alternativo. No genoma humano, mais de 70% dos miRNAs estão localizados em introns, portanto, é possível que o processo de splicing seja importante para sua maturação. O aumento da expressão do cluster de miRNAs miR-17-92 já foi relacionado ao desenvolvimento de muitas patologias, como câncer de tireoide, câncer de pulmão e linfoma. Estudos anteriores mostraram que a hnRNP A1 é importante para a maturação de do miR-18a. Neste trabalho nós investigamos a potencial associação das proteínas hnRNP A1 e hnRNP G aos miRNAs do cluster miR-17-92. Os resultados mostraram que a hnRNP A1 está associada a outros membros do cluster miR-17-92, e que a superexpressão dessa proteína aumenta as capacidades proliferativa, migratória e invasiva em células de câncer papilífero de tireóide, BCPAP.