Extending the Spacing between the Shine-Dalgarno Sequence and P-Site Codon Reduces the Rate of mRNA Translocation.

By forming base-pairing interactions with the 3' end of 16S rRNA, mRNA Shine-Dalgarno (SD) sequences positioned upstream of open reading frames facilitate translation initiation. During the elongation phase of protein synthesis, intragenic SD-like sequences stimulate ribosome frameshifting and may also slow down ribosome movement along mRNA. Here, we show that the length of the spacer between the SD sequence and P-site codon strongly affects the rate of ribosome translocation. Increasing the spacer length beyond 6 nt destabilizes mRNA-tRNA-ribosome interactions and results in a 5- to 10-fold reduction of the translocation rate. These observations suggest that during translation, the spacer between the SD sequence and P-site codon undergoes structural rearrangements, which slow down mRNA translocation and promote mRNA frameshifting.

A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation.

An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3' splice sites. U2AF1 frequently acquires an S34F-encoding mutation among patients with myelodysplastic syndromes (MDS). The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.

Observation of preQ1-II riboswitch dynamics using single-molecule FRET.

PreQ1 riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ1. Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ1-bound states of the preQ1-II riboswitch from Lactobacillus rhamnosus. We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ1 stabilize conformations that sequester the ribosome-binding site of the mRNA within the pseudoknotted structure, thus inhibiting translation initiation. Our results reveal that folding of the preQ1-II riboswitch is complex and provide evidence favoring a conformational selection model of effector binding by riboswitches of this class.

Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET.

In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5' stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31.

Single-molecule fluorescence-based studies on the dynamics, assembly and catalytic mechanism of the spliceosome.

Pre-mRNA (precursor mRNA) splicing is a key step in cellular gene expression where introns are excised and exons are ligated together to produce mature mRNA. This process is catalysed by the spliceosome, which consists of five snRNPs (small nuclear ribonucleoprotein particles) and numerous protein factors. Assembly of these snRNPs and associated proteins is a highly dynamic process, making it challenging to study the conformational rearrangements and spliceosome assembly kinetics in bulk studies. In the present review, we discuss recent studies utilizing techniques based on single-molecule detection that have helped overcome this challenge. These studies focus on the assembly dynamics and splicing kinetics in real-time, which help understanding of spliceosomal assembly and catalysis.