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.

Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways.

The selection of 3΄ splice sites (3΄ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3΄ss. Pre-mRNA molecules with two alternative 3΄ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5΄ and 3΄ sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 35.

Single-Fluorophore Detection in Femtoliter Droplets Generated by Flow Focusing.

Aqueous microdroplets with a volume of a few femtoliters are an ideal sample size for single-molecule fluorescence experiments. In particular, they enable prolonged measurements to be made on individual molecules that can diffuse freely in the surrounding medium. However, the rapid production of monodisperse droplets in a hydrodynamic flow, such as microfluidic flow focusing, will often involve volumes that are typically too large (>0.5 pL) for single-molecule studies. Desired volumes of a few femtoliters, or smaller, can be produced by either tip streaming or step emulsification in a flow-focusing device; however, in both of these methods, the aqueous droplets are dispersed in a large volume of the continuous phase, where individual droplets can diffuse perpendicular to the flow direction, and the monodispersity of droplet size produced by tip streaming is difficult to sustain for more than transient time scales. We show here that the optimized design and fabrication of microfluidic devices with shallow channel depths can result in the reliable production of stable droplets of a few femtoliters at a high rate in the dripping regime of flow focusing. Furthermore, the generated microdroplets are localized in a two-dimensional plane to enable immediate analysis. We have demonstrated the fluorescence monitoring of single molecules of encapsulated green fluorescent protein. The apparatus is straightfoward, inexpensive, and readily assembled within an ordinary laboratory environment.