40S hnRNP particles are a novel class of nuclear biomolecular condensates.

Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates

Saccharomyces cerevisiae Ngl3p is an active 3′–5′ exonuclease with a specificity towards poly-A RNA reminiscent of cellular deadenylases

Deadenylation is the first and rate-limiting step during turnover of mRNAs in eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3′–5′ exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT deadenylase complex, belonging to the DEDD and Exonuclease–Endonuclease–Phosphatase (EEP) families, respectively. Ngl3p has been identified as a new member of the EEP family of exonucleases based on sequence homology, but its activity and biological roles are presently unknown. Here, we show using in vitro deadenylation assays on defined RNA species mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3′–5′ exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p is naturally involved in processing of poly-adenylated RNA and provide insights into the mechanistic variations observed among the redundant set of EEP enzymes found in yeast and higher eukaryotes

Structural Characterization of the Saccharomyces cerevisiae THO Complex by Small-Angle X-Ray Scattering

The THO complex participates during eukaryotic mRNA biogenesis in coupling transcription to formation and nuclear export of translation-competent messenger ribonucleoprotein particles. In Saccharomyces cerevisiae, THO has been defined as a heteropentamer composed of the Tho2p, Hpr1p, Tex1p, Mft1p, and Thp2p subunits and the overall three-dimensional shape of the complex has been established by negative stain electron microscopy. Here, we use small-angle X-ray scattering measured for isolated THO components (Mft1p and Thp2p) as well as THO subcomplexes (Mft1p-Thp2p and Mft1p-Thp2p-Tho2p) to construct structural building blocks that allow positioning of each subunit within the complex. To accomplish this, the individual envelopes determined for Mft1p and Thp2p are first fitted inside those of the Mft1p-Thp2p and Mft1p-Thp2p-Tho2p complexes. Next, the ternary complex structure is placed in the context of the five-component electron microscopy structure. Our model reveals not only the position of each protein in the THO complex relative to each other, but also shows that the pentamer is likely somewhat larger than what was observed by electron microscopy

Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from <i>Clostridium difficile</i>

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Docking of THO substructures.

<p><b>A.</b> Docking of the Mft1pΔC_232-392-Thp2p heterodimer (yellow) into the larger Mft1pΔC_336-392-Thp2p heterodimer (blue). The arrows indicate the proposed position of the Mft1p C-terminus. <b>B.</b> Docking of isolated Mft1pΔC_336-392 (green) and Thp2p (red) into the larger, heterodimeric Mft1pΔC_336-392-Thp2p envelope (blue). <b>C.</b> Docking of isolated Mft1pΔC_336-392 (green) and Thp2p (red) into the smaller, heterodimeric Mft1pΔC_232-392-Thp2p envelope (yellow). <b>D.</b> Docking of the larger, dimeric Mft1pΔC_336-392-Thp2p envelope (blue) into the ternary Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597 envelope (purple). The arrows indicate the proposed position of the Tho2p C-terminus. Scale bars represent 100 Å.</p

Placement of subunits in the THO complex.

<p><b>A.</b> Model showing the proposed location and orientation of the subunits Mft1p (green), Thp2p (red), and Tho2p (blue) within the ternary Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597 THO complex (purple). The proposed location of the N and C terminal regions of Tho2p and Mft1p are indicated. Scale bars represent 100 Å. <b>B.</b> Comparison of the ternary Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597 SAXS envelope with the five-component EM reconstruction of the THO complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103470#pone.0103470-Pena1" target="_blank">[13]</a>. The positions of the proteins not part of our sample (Hpr1p and Tex1p) are indicated.</p

Purification of THO subcomplexes.

<p><b>A.</b> Overview of the Tho2p, Mft1p and Thp2p constructs used relative to their full-length forms (boxed in grey). Blue boxes: Stretches of residues removed to obtain the construct directly below. Purple boxes: Strep II tag. <b>B.</b> THO complexes (THO1, 3, 4, and 5) were expressed and purified from <i>E. coli</i> in two steps and analysed by Coomassie-stained SDS-PAGE. The table shows for each construct the molecular weight of each protein and any associated tags (H = 6xHis, S = Strep II) as well as whether the protein is expressed (+/-). Positions on the gel for proteins confirmed by mass spectrometry are indicated with arrowheads and * indicates an <i>E. coli</i> protein contaminant. <b>C.</b> Purified THO subcomplexes analysed by Coomassie-stained SDS-PAGE: Heterodimeric THO4 (Mft1pΔC_270-392-Thp2p), heterodimeric THO3 (Mft1pΔC_336-392-Thp2p), and heterotrimeric THO3 (Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597). <b>D.</b> Overlay of gel filtration chromatograms obtained during isolation of the ternary Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597 (Trimer) from the binary Mft1pΔC_336-392-Thp2p (Dimer). Between runs 1 (blue), 2, (red), and 3 (green), peak fractions were pooled, concentrated and re-applied to the column. Elution retention volumes are noted along with the positions of standards used for calibration: Blue Dextran (V₀, void volume, 2000 kDa), ferritin (440 kDa), aldolase (158 kDa), and conalbumin (75 kDa). Units on the y-axis are mAU absorption at 280 nm.</p

Envelopes of THO proteins and complexes obtained by SAXS.

<p>Molecular envelopes obtained using DAMMIF for THO proteins and subcomplexes. <b>A</b>. Mft1pΔC_336-392 (as isolated from the homodimer). <b>B</b>. Thp2p (as isolated from the homodimer). <b>C</b>. Mft1pΔC_232-392-Thp2p. <b>D</b>. Mft1pΔC_336-392-Thp2p. <b>E</b>. Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597. The proteins and complexes are each represented with an envelope shown in three perpendicular views as indicated. Scale bars represent 100 Å.</p

An in vitro reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands

U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon–intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.ISSN:1362-4962ISSN:0301-561