Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.

We have recently identified an intronic polymorphic CA-repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA-repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA-rich clusters. Therefore, we have systematically searched the human genome databases for CA-repeat and CA-rich elements associated with alternative 5' splice sites (5'ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5'ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.

We give a description of a Petri net-based framework for modelling and analysing biochemical pathways, which unifies the qualitative, stochastic and continuous paradigms. Each perspective adds its contribution to the understanding of the system, thus the three approaches do not compete, but complement each other. We illustrate our approach by applying it to an extended model of the three stage cascade, which forms the core of the ERK signal transduction pathway. Consequently our focus is on transient behaviour analysis. We demonstrate how qualitative descriptions are abstractions over stochastic or continuous descriptions, and show that the stochastic and continuous models approximate each other. Although our framework is based on Petri nets, it can be applied more widely to other formalisms which are used to model and analyse biochemical networks. MotivationBiochemical reaction systems have by their very nature three distinctive characteristics.(1) They are inherently bipartite, i.e. they consist of two types of game players, the species and their interactions. (2) They are inherently concurrent, i.e. several interactions can usually happen independently and in parallel. (3) They are inherently stochastic, i.e. the timing behaviour of the interactions is governed by stochastic laws. So it seems to be a natural choice to model and analyse them with a formal method, which shares exactly these distinctive characteristics: stochastic Petri nets.However, due to the computational efforts required to analyse stochastic models, two abstractions are more popular: qualitative models, abstracting away from any time dependencies, and continuous models, commonly used to approximate stochastic behaviour by a deterministic one. We describe an overall framework to unify these three paradigms, providing a family of related models with high analytical power.The advantages of using Petri nets as a kind of umbrella formalism are seen in the following: M. Bernardo, P. Degano, and G. Zavattaro (Eds.): SFM

Alternative mRNA splicing patterns are determined by the combinatorial control of regulator proteins and their target RNA sequences. We have recently characterized human hnRNP L as a global regulator of alternative splicing, binding to diverse C/Arich elements. To systematically identify hnRNP L target genes on a genome-wide level, we have combined splice-sensitive microarray analysis and an RNAi-knockdown approach. As a result, we describe 11 target genes of hnRNP L that were validated by RT-PCR and that represent several new modes of hnRNP L-dependent splicing regulation, involving both activator and repressor functions: first, intron retention; second, inclusion or skipping of cassette-type exons; third, suppression of multiple exons; and fourth, alternative poly(A) site selection. In sum, this approach revealed a surprising diversity of splicing-regulatory processes as well as poly(A) site selection in which hnRNP L is involved.