The Protein Data Bank (PDB)--the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes--was established in 1971, becoming the first open-access digital resource in the biological sciences The PDB archive currently houses ~130,000 entries (May 2017) It is managed by the Worldwide Protein Data Bank organization (wwPDB; wwpdborg), which includes the RCSB Protein Data Bank (RCSB PDB; rcsborg), the Protein Data Bank Japan (PDBj; pdbjorg), the Protein Data Bank in Europe (PDBe; pdbeorg), and BioMagResBank (BMRB; wwwbmrbwiscedu) The four wwPDB partners operate a unified global software system that enforces community-agreed data standards and supports data Deposition, Biocuration, and Validation of ~11,000 new PDB entries annually (depositwwpdborg) The RCSB PDB currently acts as the archive keeper, ensuring disaster recovery of PDB data and coordinating weekly updates wwPDB partners disseminate the same archival data from multiple FTP sites, while operating complementary websites that provide their own views of PDB data with selected value-added information and links to related data resources At present, the PDB archives experimental data, associated metadata, and 3D-atomic level structural models derived from three well-established methods: crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM) wwPDB partners are working closely with experts in related experimental areas (small-angle scattering, chemical cross-linking/mass spectrometry, Forster energy resonance transfer or FRET, etc) to establish a federation of data resources that will support sustainable archiving and validation of 3D structural models and experimental data derived from integrative or hybrid methods

Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive.

Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3' region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5' splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5' splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem-pre-mRNA and Brr2-U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation.

Structure of a pre-catalytic spliceosome

RNAComposer is a fully automated, web-interfaced system for RNA 3D structure prediction, freely available at http://rnacomposer.cs.put.poznan.pl/ and http://rnacomposer.ibch.poznan.pl/. Its main components are: manually curated database of RNA 3D structure elements, highly efficient computational engine and user-friendly web application. In this paper, we demonstrate how the latest additions to the system allow the user to significantly affect the process of 3D model composition on several computational levels. Although in general our method is based on the knowledge of secondary structure topology, currently the RNAComposer offers a choice of six incorporated programs for secondary structure prediction. It also allows to apply a conditional search in the database of 3D structure elements and introduce user-provided elements into the final 3D model. This new functionality contributes to a significant improvement of the predicted 3D model reliability and it facilitates a better model adjustment to the experimental data. This is exemplified based on the RNAComposer application for modelling of the 3D structures of precursors of the miR160 family members.

/pdf/new-functionality-of-rnacomposer-an-application-to-shape-the-3r8239rqe5.pdf

New functionality of RNAComposer: an application to shape the axis of miR160 precursor structure.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors1,2. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements3. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages4, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis6-8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.

Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

Multiparametric high-resolution imaging of native proteins by force-distance curve–based AFM

Multiparametric high-resolution imaging of native proteins by force-distance curve-based AFM.

Transcription Increases the Cooperativity of Ribonucleoprotein Assembly

Structural Analysis of Multi-Helical RNAs by NMR–SAXS/WAXS: Application to the U4/U6 di-snRNA

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation.

/pdf/single-molecule-analysis-reveals-reversible-and-irreversible-6tppk90ybe.pdf

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

Many of the cell's macromolecular machines contain multiple components that transiently associate with one another. This compositional and dynamic complexity presents a challenge for understanding how these machines are constructed and function. Colocalization single molecule spectroscopy enables simultaneous observation of individual components of these machines in real-time and grants a unique window into processes that are typically obscured in ensemble assays. Colocalization experiments can yield valuable information about assembly pathways, compositional heterogeneity, and kinetics that together contribute to the development of richly detailed reaction mechanisms. This review focuses on recent advances in colocalization single molecule spectroscopy and how this technique has been applied to enhance our understanding of transcription, RNA splicing, and translation.

Visualizing cellular machines with colocalization single molecule microscopy

The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Forster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg(2+) and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg(2+) and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg(2+) may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg(2+) or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg(2+) toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg(2+)-dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.

/pdf/conformational-dynamics-of-stem-ii-of-the-u2-snrna-4y2oecsc4b.pdf

Margaret L. Rodgers

Papers

Transcription Increases the Cooperativity of Ribonucleoprotein Assembly

Structural Analysis of Multi-Helical RNAs by NMR–SAXS/WAXS: Application to the U4/U6 di-snRNA

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

Visualizing cellular machines with colocalization single molecule microscopy

Conformational dynamics of stem II of the U2 snRNA.