Genomewide RNA binding of hnRNP L monitored by iCLIP: new role in noncoding RNA biogenesis?

We have characterized human hnRNP L as a global regulator of alternative splicing binding to CA-repeat and CA-rich elements. Computational searches and a combined RNAi/microarray-approach revealed several target genes whose splicing patterns are regulated by hnRNP L [1-3].

We currently use a novel genomewide approach, termed iCLIP (individual nucleotide resolution UV-crosslinking/immunoprecipitation, in collaboration with Jernej Ule, Cambridge, UK), which allows global insight into in vivo binding targets of hnRNP L. HeLa cells are irradiated with UV light, inducing protein-RNA crosslinks. After immunoprecipitation of hnRNP L-RNA complexes, the RNA tags are purified, amplified by RT-PCR and subjected to Solexa high-throughput sequencing, revealing all in vivo binding targets. Sequence analysis displays the binding characteristics of the protein and its function in RNA metabolism and alternative splicing regulation [4].

Initial results suggest that hnRNP L plays a new role in the biogenesis of noncoding RNAs, since we observe dense clustering of iCLIP tags in introns that harbor snoRNAs and microRNAs, for instance snoRNA-containing introns in the EIF4A2, MATR3, RCC1 and GAS5 genes. In addition, a non-coding 3’-extension of the TAF1D gene, which harbors 7 snoRNAs and one miRNA, is generated by suppression of the major poly(A) site, regulated by hnRNP L. Finally, we were able to validate clustered hnRNP L binding in the long noncoding MALAT1 RNA.