Aberration-corrected electron microscopy of nanoparticles

The early history of scanning transmission electron microscopy (STEM) is reviewed as a way to frame the technical issues that make aberration correction an essential upgrade for the study of nanoparticles using STEM. The principles of aberration correction are explained, and the use of aberration-corrected microscopy in the study of nanostructures is exemplified in order to remark the features and challenges in the use of this measuring techniqu

The group II intron ribonucleoprotein precursor is a large, loosely packed structure

Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

GFP-tagged snRNP components reveal the dynamics and rate for spliceosome assembly in vivo

The 5' untranslated region of the serotonin receptor 2C pre-mRNA generates miRNAs and is expressed in non-neuronal cells

The serotonin receptor 2C (HTR2C) gene encodes a G protein-coupled receptor that is exclusively expressed in neurons. Here, we report that the 5' untranslated region of the receptor pre-mRNA as well as its hosted miRNAs is widely expressed in non-neuronal cell lines. Alternative splicing of HTR2C is regulated by MBII-52. MBII-52 and the neighboring MBII-85 cluster are absent in people with Prader-Willi syndrome, which likely causes the disease. We show that MBII-52 and MBII-85 increase expression of the HTR2C 5' UTR and influence expression of the hosted miRNAs. The data indicate that the transcriptional unit expressing HTR2C is more complex than previously recognized and likely deregulated in Prader-Willi syndrome.This work was supported by NIH RO1 GM083187, P20RR020171 to SS; GM079549 to RS and JS; Binational Science Foundation (BSF), USA-Israel, transformative Grant, #2010508, to SS and RS. EE and AP were supported by the Spanish Ministry of Science with grant BIO2011-23920 and by Sandra Ibarra Foundation for Cancer with grant FSI 2011-03

Structural characterization of the fission yeast U5.U2/U6 spliceosome complex

The spliceosome is a dynamic macromolecular machine that catalyzes the excision of introns from pre-mRNA. The megadalton-sized spliceosome is composed of four small nuclear RNPs and additional pre-mRNA splicing factors. The formation of an active spliceosome involves a series of regulated steps that requires the assembly and disassembly of large multiprotein/RNA complexes. The dynamic nature of the pre-mRNA splicing reaction has hampered progress in analyzing the structure of spliceosomal complexes. We have used cryo-electron microscopy to produce a 29-Å density map of a stable 37S spliceosomal complex from the genetically tractable fission yeast, Schizosaccharomyces pombe. Containing the U2, U5, and U6 snRNAs, pre-mRNA splicing intermediates, U2 and U5 snRNP proteins, the Nineteen Complex (NTC), and second-step splicing factors, this complex closely resembles in vitro purified mammalian C complex. The density map reveals an asymmetric particle, ≈30 × 20 × 18 nm in size, which is composed of distinct domains that contact each other at the center of the complex