Liver RBFOX2 regulates cholesterol homeostasis via Scarb1 alternative splicing in mice

RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases

The Involvement of SRSF1 in pre-mRNA splicing

The key splicing signals in pre-mRNA, the branch-point, 5’ splice site and 3’ splice site, are exceptionally poorly conserved in mammals. To compensate for this, key regulatory sequences are required to direct the reciprocal factors, U2AF, U1 snRNP and U2 snRNP respectively, to the correct sites. These regulatory sequences function by recruiting activators, of which the main family are the SR proteins, or repressors, of which the main family are the hnRNP proteins, which in turn stimulate or repress the binding of key spliceosomal factors. The archetypal SR protein is SRSF1. SRSF1 was the first non-snRNP factor identified and the first found to control alternative splicing. Its best-understood activity is to stimulate the inclusion of exons by binding to purine-rich exonic splicing enhancer (ESE) sequences. There is also some evidence suggesting its involvement in constitutive splicing, which began with demonstrations that it could compensate for depletion of the U1 snRNP. However, further investigations into both its recruitment via ESEs and its possible role in constitutive splicing have foundered due to apparent nonstoichiometric binding. Single molecule experiments allow us to look at the exact number of SRSF1 proteins that bind. The experiments outlined here indicate that the U1 snRNP can actually recruit SRSF1 in a stoichiometric manner. This implicates a possible recruitment mechanism for SRSF1 which would allow it to play a role in core splicing reactions and exon definition. Furthermore we demonstrate that with increasing numbers of enhancers, which sequentially increase splicing efficiency, the number of SRSF1 proteins bound does not change but the chance of a protein binding event increases. This fits a model in which the initial binding of SRSF1 is weak and transient. The same construct is also used to show that introducing a non RNA link in between an ESE and its target site does not silence the ESEs effect, indicating that ESEs exert their effect via RNA loops

The mechanisms of a mammalian splicing enhancer.

Exonic splicing enhancer (ESE) sequences are bound by serine & arginine-rich (SR) proteins, which in turn enhance the recruitment of splicing factors. It was inferred from measurements of splicing around twenty years ago that Drosophila doublesex ESEs are bound stably by SR proteins, and that the bound proteins interact directly but with low probability with their targets. However, it has not been possible with conventional methods to demonstrate whether mammalian ESEs behave likewise. Using single molecule multi-colour colocalization methods to study SRSF1-dependent ESEs, we have found that that the proportion of RNA molecules bound by SRSF1 increases with the number of ESE repeats, but only a single molecule of SRSF1 is bound. We conclude that initial interactions between SRSF1 and an ESE are weak and transient, and that these limit the activity of a mammalian ESE. We tested whether the activation step involves the propagation of proteins along the RNA or direct interactions with 3' splice site components by inserting hexaethylene glycol or abasic RNA between the ESE and the target 3' splice site. These insertions did not block activation, and we conclude that the activation step involves direct interactions. These results support a model in which regulatory proteins bind transiently and in dynamic competition, with the result that each ESE in an exon contributes independently to the probability that an activator protein is bound and in close proximity to a splice site

Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5 ' splice site (SS), and both factors affect 5 ' SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5 ' SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.ISSN:0261-4189ISSN:1460-207

Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5′ splice site (SS), and both factors affect 5′ SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5′SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals

From the bottom up: dimensional control and characterization in molecular monolayers

Self-assembled monolayers are a unique class of nanostructured materials, with properties determined by their molecular lattice structures, as well as the interfaces with their substrates and environments. As with other nanostructured materials, defects and dimensionality play important roles in the physical, chemical, and biological properties of the monolayers. In this review, we discuss monolayer structures ranging from surfaces (two-dimensional) down to single molecules (zero-dimensional), with a focus on applications of each type of structure, and on techniques that enable characterization of monolayer physical properties down to the single-molecule scale