Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

<div><p>Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11–48K and U11/U12–65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5′ splice sites (5′ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25–50 nt downstream from the target 3′ splice site (3′ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11–35K protein for 3′ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5′ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5′ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.</p></div

Crystal Structure of an Engineered LRRTM2 Synaptic Adhesion Molecule and a Model for Neurexin Binding

Synaptic adhesion molecules are key components in development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of chemical synapses. Several families of neuronal adhesion molecules have been identified such as the neuronal cell adhesion molecules, neurexins and neuroligins, and in particular recently several leucine-rich repeat proteins, e.g., Netrin G-ligands, SLITRKs, and LRRTMs. The LRRTMs form a family of four proteins. They have been implicated in excitatory glutamatergic synapse function and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. In addition, LRRTM3 and LRRTM4 have been found to be ligands for heparan sulfate proteoglycans, including glypican. We report here the crystal structure of a thermostabilized mouse LRRTM2, with a <i>T</i>_m 30 °C higher than that of the wild-type protein. We localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation, and prior information about the interaction of the ligand with neurexins, which allowed us to propose a tentative model for the LRRTM–neurexin interaction complex. We also determined affinities of the thermostabilized LRRTM2 and wild-type LRRTM1 and LRRTM2 for neurexin-β1 with and without Ca²⁺. Cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapselike contacts. The structural and functional data presented here provide the first structure of an LRRTM protein and allow us to propose a model for the molecular mechanism of LRRTM function in the synaptic adhesion