Biochemistry

iCLIP at nucleotide resolution

Most, if not all, human mRNAs are spliced into several isoforms by heterogeneous nuclear ribonucleoprotein (hnRNP) particles that associate with the nascent transcript. Koenig et al. now precisely map binding of an hnRNP by a twist on UV-light cross-linking and immunoprecipitation (CLIP). In individual-nucleotide-resolution CLIP (iCLIP), each RNA reveals the exact site of protein binding. These data will yield insight into the effect of hnRNP binding on splicing.

Koenig, J. et al. Nat. Struct. Mol. Biol. 7, 909–915 (2010).

Proteomics

O-glycosylation stoichiometry

Sugar-based post-translational modifications remain a challenge to characterize, owing to their low abundance and diverse chemistries. Rexach et al. now describe a straightforward approach to characterize the stoichiometry and dynamics of O-GlcNAc glycosylation in vivo, by selectively labeling terminal O-GlcNAc sugars with a polyethylene glycol–based mass tag that can be easily detected by denaturing polyacrylamide electrophoresis and immunoblotting. This allowed them to follow the interplay of phosphorylation and glycosylation in mammalian cells.

Rexach, J.E. et al. Nat. Chem. Biol. advance online publication (25 July 2010).

Stem cells

Changing substrate rigidity to grow stem cells

There is increasing interest in understanding how mechanical properties of the growth substrate influence cellular biology. Gilbert et al. now show that muscle stem cells can be cultured for one week on hydrogel surfaces with a rigidity that matches that of muscle. In contrast to rigid surfaces, the soft surface promotes stem cell self-renewal and maintains the ability of the cells to regenerate muscle when transplanted in vivo. Whether the effect persists for culture over longer periods awaits further study.

Gilbert, P.M. Science advance online publication (15 July 2010).

Microscopy

tRNA movement through the ribosome

Time-resolved, single-particle electron cryomicroscopy allows biological processes to be followed by taking multiple snapshots of dynamic systems, but a very large computational effort is required to sort images of heterogeneous complexes. By taking and analyzing two million images of tRNA movement through the ribosome at various time points, Fischer et al. used this technique to observe 50 distinct ribosome sub-states, gaining insights into the mechanism of the translocation step of protein synthesis.

Fischer, N. et al. Nature 466, 329–333 (2010).

Proteomics

Characterizing protein ubiquitination

The low abundance of ubiquitination sites make them difficult to detect. Xu et al. now describe a method to enrich these previously ubiquitinated peptides with a monoclonal antibody reagent that recognizes a unique diglycine adduct left on modified lysine residues after tryptic digestion. Using mass spectrometry, they identified 374 ubiquitination sites on 236 human proteins, adding substantially to the number of known ubiquitination sites.

Xu, G. et al. Nat. Biotechnol. advance online publication (18 July 2010).