The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an abundant nuclear RNA-binding protein, plays both nuclear and cytoplasmic roles in mRNA export of intronless genes, IRES-mediated translation, mRNA stability and alternative splicing regulation. Recently, it was reported that hnRNP L autoregulates its own expression on the level of alternative splicing. HnRNP L protein recognises CA-repeat as well as CA-rich clusters. HnRNP L-like (LL) protein is a paralog of hnRNP L, whose expression is upregulated in a tissue-specific manner. It was shown that hnRNP LL regulates alternative splicing of CD45 exon 4 upon T-cell activation.HnRNP L and hnRNP LL share 58% overall amino acid identity and have similar sizes (558 vs. 542 amino acids). Both proteins contain four classical RNA recognition motifs (RRMs). The glycine-rich region of hnRNP L is less pronounced in hnRNP LL, and proline-rich region of hnRNP L is absent in hnRNP LL.To investigate the role of individual domains in hnRNP L and hnRNP LL protein function, I created a series of deletion derivatives and mutants with one or several amino acid substitutions in individual RNA-binding domains.First, I analysed the RNA-binding properties of the full-length and deletion constructs of hnRNP L and LL by EMSA (electrophoretic mobility shift assay) and filter binding assay. Two substrates were used: CA-repeat and CA-rich RNAs. I demonstrated that the combination of two RNA-binding domains of hnRNP L (RRMs 1/2 and 2/3) is both necessary and sufficient for high-affinity binding to CA-repeat RNA. In contrast, the high-affinity binding of hnRNP L to CA-rich RNA requires all four RRMs. In the case of hnRNP LL all four RRM domains are required for high-affinity binding to both substrates. Mutation analysis revealed that hnRNP L RRM2 is a major determinant for RNA binding specificity and affinity.EMSA in combination with gel filtration of protein-RNA complexes indicated that hnRNP L requires at least two high-score binding motifs, separated by a short spacer (7-10 nucleotides) to bind tightly to the RNA.Second, hnRNP L mutant derivatives were tested for alternative splicing activity, using a SLC2A2 minigene construct and hnRNP L depleted nuclear extract. I demonstrated that only full-length protein and not the truncated mutant proteins could function as a repressor in regulation of alternative splicing.In addition, a specific role of inter-domain interaction between RRMs 3 and 4 in CA-rich RNA binding and function of hnRNP L as a splicing repressor was uncovered.In sum, my results suggest that the presence of all four RRMs is essential for splicing repressor activity of hnRNP L, whereas two RRMs are sufficient for tight associationwith CA-repeat RNA
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