We uncovered the diversity of non-canonical splice sites at the human transcriptome using deep transcriptome profiling. We mapped a total of 3.7 billion human RNA-seq reads and developed a set of stringent filters to avoid false non-canonical splice site detections. We identified 184 splice sites with non-canonical dinucleotides and U2/U12-like consensus sequences. We selected 10 of the herein identified U2/U12-like non-canonical splice site events and successfully validated 9 of them via reverse transcriptase-polymerase chain reaction and Sanger sequencing. Analyses of the 184 U2/U12-like non-canonical splice sites indicate that 51% of them are not annotated in GENCODE. In addition, 28% of them are conserved in mouse and 76% are involved in alternative splicing events, some of them with tissue-specific alternative splicing patterns. Interestingly, our analysis identified some U2/U12-like non-canonical splice sites that are converted into canonical splice sites by RNA A-to-I editing. Moreover, the U2/U12-like non-canonical splice sites have a differential distribution of splicing regulatory sequences, which may contribute to their recognition and regulation. Our analysis provides a high-confidence group of U2/U12-like non-canonical splice sites, which exhibit distinctive features among the total human splice sites.

Corticotropin releasing factor binding protein (CRF-BP) was originally recognized as CRF sequestering protein. However, its differential subcellular localization in different brain nuclei suggests that CRF-BP may have additional functions. There is evidence that CRF-BP potentiates CRF and urocortin 1 actions through CRF type 2 receptors (CRF 2 R). CRF 2 R is a G protein-coupled receptor (GPCR) that is found mainly intracellularly as most GPCRs. The access of GPCRs to the cell surface is tightly regulated by escort proteins. We hypothesized that CRF-BP binds to CRF 2 R, exerting an escort protein role. We analyzed the colocalization of CRF-BP and CRF 2 R in cultured rat mesencephalic neurons, and the localization and interaction of heterologous expressed CRF-BP and CRF 2α R in yeast, human embryonic kidney 293, and rat pheochromocytoma 12 cells. Our results showed that CRF-BP and CRF 2 R naturally colocalize in the neurites of cultured mesencephalic neurons. Heterologous expression of each protein showed that CRF-BP was localized mainly in secretory granules and CRF 2α R in the endoplasmic reticulum. In contrast, CRF-BP and CRF 2α R colocalized when both proteins are coexpressed. Here we show that CRF-BP physically interacts with the CRF 2α R but not the CRF 2β R isoform, increasing CRF 2α R on the cell surface. Thus, CRF-BP emerges as a GPCR escort protein increasing the understanding of GPCR trafficking.T he corticotropin releasing factor (CRF) system plays a key role in the response and adaptation to stressful stimuli (1, 2) and in the interaction between stress and addiction (3). The CRF system acts on the hypothalamic-pituitary-adrenal axis (4, 5) and in different brain regions (1, 6). The CRF system includes four peptides, CRF type 1 (CRF 1 R) and type 2 (CRF 2 R), G protein-coupled receptors (GPCRs) (6-8), and CRF binding protein (CRF-BP) (9). CRF-BP was described as a circulating polypeptide in pregnant women (10, 11). CRF-BP binds CRF and urocortin with high affinity (12). Different functions have been proposed for CRF-BP (13). On one hand, CRF-BP exerts an inhibitory role by sequestering CRF peptide (9, 14-16). On other hand, a facilitatory role of CRF-BP on CRF-dependent neuronal plasticity depending on CRF 2 R in the rat ventral tegmental area (VTA) has been described (17, 18). Recently, it has been shown that CRF-BP and CRF 2 R are important for ethanol binge drinking behavior (19). The anatomical evidence showing that CRF-BP has different subcellular distribution depending on the neuronal context (20) further supports several roles for CRF-BP.Three isoforms of CRF 2 R have been reported, the α isoform being the most expressed in the brain (21). CRF 2α R is localized intracellularly in neurons of the rat dorsal raphe nucleus and that exposure to acute (22) and repeated stress (23) increases its presence in the plasma membrane. CRF 2α R overexpressed in human embryonic kidney (HEK293T) cells is associated with the endoplasmic reticulum (ER) (24). Schulz et al. (25) showed that CRF 2α R is retained in...

The lateral septum (LS), a brain structure implicated in addictive behaviours, regulates the activation of dopaminergic neurones in the ventral tegmental area. Vasopressinergic projections from the extended amygdala to the LS, which are sexually dimorphic, could be responsible for the vulnerability to addiction in a sex-dependent manner. The present study aimed to investigate the modulatory effects of amphetamine (AMPH) on the expression of vasopressin (AVP) in the vasopressinergic extra-hypothalamic system in sensitised male and female rats. Adult male and female Sprague-Dawley rats underwent an AMPH-locomotor sensitisation protocol. Acute AMPH increased AVP mRNA expression in the medial amygdala (MeA), whereas AMPH-induced sensitisation increased AVP mRNA expression in the bed nucleus of the stria terminalis (BNST) only in females. Interestingly, the increase in AVP expression in BNST was higher in oestrus females compared to dioestrus females and acute AMPH resulted in a decrease in AVP levels in the LS, only in males. Thus, there are complex and region-specific interactions between AMPH and the extra-hypothalamic vasopressinergic system in the brain, underlying possible alterations in different behaviours caused by acute and chronic AMPH exposure.