Megan Mayerle

Protein-guided RNA dynamics during early ribosome assembly.

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4–RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein–RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA–protein complexes.

Assembly of the five-way junction in the ribosomal small subunit using hybrid MD-Gō simulations

Assembly of the bacterial ribosomal small subunit (SSU) begins with the folding of the five-way junction upon interaction with the primary binding protein S4. This complex contains the largest contiguous molecular signature, which is a conserved feature in all bacterial 16S rRNAs. In a previous study, we used all-atom molecular dynamics simulations to demonstrate that the co-evolving signature in the N-terminus of S4 is intrinsically disordered and capable of accelerating the binding process through a fly casting mechanism. In this paper, comparisons between the all-atom MD simulations and FRET experiments identify multiple metastable conformations of the naked five-way junction without the presence of S4. Furthermore, we capture the simultaneous folding and binding of the five-way junction and r-protein S4 by use of a structure-based Gō potential implemented within the framework of the all-atom molecular dynamics CHARMM force field. Different folding pathways are observed for the refolding of the five-way junction upon partial binding of S4. Our simulations illustrate the complex nature of RNA folding in the presence of a protein binding partner and provide insight into the role of population shift and the induced fit mechanisms in the protein:RNA folding and binding process.

Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency

Significance The spliceosome, which catalyzes pre-mRNA splicing via a two-step process, must balance the need for high-fidelity splice-site selection with the need for rapid, efficient splicing. We propose that the RNaseH domain (RH) of Prp8 contributes to this balance by toggling between two different conformations throughout the splicing cycle. Using a set of previously published prp8 alleles, we link alleles that stabilize one conformation of RH to high-fidelity, low-efficiency splicing and those that stabilize the other to low-fidelity, high-efficiency splicing. This model is consistent with recent data that indicate the conformation of the spliceosome is similar at both catalytic steps and provides an example of a structural basis for splicing fidelity. Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the “master regulator” of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome’s catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome “toggles” between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

Genetics and biochemistry remain essential in the structural era of the spliceosome

The spliceosome is not a single macromolecular machine. Rather it is a collection of dynamic heterogeneous subcomplexes that rapidly interconvert throughout the course of a typical splicing cycle. Because of this, for many years the only high resolution structures of the spliceosome available were of smaller, isolated protein or RNA components. Consequently much of our current understanding of the spliceosome derives from biochemical and genetic techniques. Now with the publication of multiple, high resolution structures of the spliceosome, some question the relevance of traditional biochemical and genetic techniques to the splicing field. We argue such techniques are not only relevant, but vital for an in depth mechanistic understanding of pre-mRNA splicing.

Brr2 is a splicing fidelity factor

Many spliceosomal DExD/H box helicases act as fidelity factors during pre-mRNA splicing, promoting on-pathway interactions while simultaneously minimizing errors. Mutations linked to Retinitis Pigmentosa (RP), a form of heritable blindness, map to key domains of spliceosomal helicase Brr2 (SNRNP200 in humans). Previous data show that such mutations negatively impact spliceosome activation, likely due to defects in brr2-RP RNA binding, helicase, and ATPase activities. Furthermore, data from human reporter constructs suggest that brr2-RP might impact 5′ splice site selection. Here we undertake a systematic analysis of brr2-RP effects on splicing fidelity. We show that a subset of brr2-RP mutants exhibit intron retention in vivo. Furthermore, brr2-RP mutants display hyperaccurate and/or error-prone splicing of a variety of splicing reporters. Branch-site fidelity is particularly impacted in this reporter assay. In addition, multiple brr2-RP alleles genetically interact with prp16 alleles known to impact the fidelity of branch site selection. Together these data implicate Brr2 in the fidelity of branch-site selection, and suggest that RP results not just from defects in spliceosome activation, but also from fidelity defects arising throughout the splicing cycle and in splicing fidelity.

A new communication hub in the RNA world

During assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), the RNA-binding protein (RBP) Gemin5 recognizes the snRNP code and interacts with the large Gemin2–SMN complex. So et al. now find that Gemin2 also interacts with U1-70K, thereby conferring a preferential advantage on U1 snRNP assembly, and they extrapolate that SMN–Gemin2 serves a general ribonucleoprotein-exchange function.