Volume 20 Issue 6, June 2013

Whether centromere-specific CENP-A–containing nucleosomes comprise one molecule each of CENP-A and histones H4, H2A and H2B (forming a tetramer or hemisome) or two molecules of all four histones (forming an octamer) has been controversial. Three new studies now address this controversy using complementary in vitro and in vivo approaches.

Eukaryotic mRNAs containing nonsense codons are degraded by nonsense-mediated mRNA decay (NMD). In humans, NMD was proposed to act exclusively on newly synthesized mRNA during a 'pioneer round' of translation, when mRNA is associated with the nuclear cap-binding complex (CBC). Two reports now challenge this view, revealing that NMD is independent of whether CBC or the cytoplasmic cap-binding protein (eIF4E) promotes translation and can occur later in an mRNA's life.

At last, structures have been solved for different conformational states, facing in and facing out, for the same transporter protein of the major facilitator superfamily. The structural basis of alternating substrate access for binding and translocation can now be properly visualized.

Whereas post-translational modifications (PTMs) in histone tails are well characterized, functional and mechanistic insights into PTMs in the globular nucleosome core have been lacking. This Perspective covers recent advances in the understanding of lateral-surface PTMs and their effects on nucleosome and chromatin dynamics.

Human Dicer can process long double-stranded RNA and hairpin precursor RNA to yield short interfering RNAs or microRNAs, respectively. EM and single-particle analyses of Dicer–substrate complexes now provide insight into the structural basis of Dicer's substrate preference, implicating RNA structure and cofactors in determining substrate recognition and processing efficiency by Dicer.

The telomere position effect (TPE) can change gene expression at intermediate telomere lengths in cultured human cells, but it has not been previously implicated in disease pathogenesis. A gene implicated in facioscapulohumeral muscular dystrophy (FSHD) pathogenesis, and located adjacent to a chromosome end, is now shown to be upregulated in muscle cells with short telomeres, which suggests that TPE contributes to the late-onset phenotype of FSHD.

Interactions between synaptotagmin-1 and the SNARE syntaxin-1 are known to mediate synaptic-vesicle exocytosis. Fusion experiments with artificial lipid membranes combined with the crystal structure of synaptotagmin's C2B domain bound to phosphoserine indicate that PIP2 clusters, organized by syntaxin, act as molecular beacons for vesicle docking and direct Ca²⁺-dependent membrane fusion.

Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location, but the histone composition and stoichiometry of CENP-A nucleosomes have been controversial. ChIP-seq and biochemical analyses of in vitro–reconstituted CENP-A nucleosomes now demonstrate that the predominant form at functional centromeres is an octamer with loose DNA ends.

HECT E3 ligases directly catalyze ubiquitin transfer to their substrates. The crystal structure of ubiquitin-loaded Nedd4 HECT domain in complex with ubiquitin is now presented, revealing insights into the mechanisms for catalytic priming and sequential addition and suggesting the basis for Lys63 specificity.

It has been suggested that nonsense-mediated mRNA decay (NMD) is restricted to a pioneer round of translation initiated on mRNAs associated with the cap-binding complex (CBC), whereas the exchange of the CBC for the eIF4F translation initiation complex renders mRNAs immune to NMD. However, multiple lines of evidence now suggest that eIF4F-associated mRNAs are not immune to NMD.

It has been suggested that nonsense-mediated mRNA decay (NMD) is restricted to cap-binding complex (CBC)-bound mRNAs during the pioneer round of translation in mammalian cells. However, analysis of the decay kinetics of mRNAs bound to either CBC or the eukaryotic initiation factor 4E in human cells shows that both substrate types are equally susceptible to NMD.

During translation, elongation factor G (EF-G) and transfer RNAs alternately bind the ribosome to direct protein synthesis. Using single-molecule fluorescence with zero-mode waveguide, EF-G–GTP is shown to continuously sample both rotational states of the ribosome, binding with higher affinity to the rotated state to stimulate translocation and return to the nonrotated state.

The core of the spliceosome contains PRP8, a highly conserved protein that contacts each of the active site components and is implicated in both steps of pre-mRNA splicing. New work shows that structural rearrangement of the PRP8 RNase H domain between the first and second steps forms a Mg²⁺-binding site that is required for the second splicing reaction, revealing the molecular basis of the two-state model of splicing catalysis.

Tristetraprolin (TTP) post-transcriptionally represses gene expression by interacting with AU-rich elements in 3A untranslated regions of target mRNAs and mediates deadenylation and decay by recruiting the CCR4–NOT deadenylase complex through the CNOT1 subunit. Structural and mutational studies now show how the TTP-CNOT1 interaction brings about TTP-mediated deadenylation of target mRNAs.

The bacterial metalloregulator IscR possesses the unusual ability to direct differential gene expression via specific recognition of two distinct operator motifs in an Fe-S–dependent manner. New work shows that Fe-S binding displaces a residue required for sequence discrimination at the DNA binding interface, revealing a conditional, ligand-mediated mechanism regulating sequence-specific recognition.

Genetic evidence has implicated the SOS-induced translesion DNA polymerase DinB (Pol IV) in error-prone recombination. Now biochemical analyses show that Pol IV outcompetes high-fidelity polymerases during the extension of D-loop recombination intermediates under SOS response conditions, providing new insight into the mechanism of stress-induced mutations in Escherichia coli.

Tau is a microbutule-associated protein that can form pathological aggregates in neurodegenerative conditions such as Alzheimer's disease. Tau can undergo acetylation at lysine residues within its microtubule-binding repeats, a modification that impairs tau function and promotes its aggregation. Now tau is identified as an acetyltransferase able to modify itself.

Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location. Their reduced height relative to canonical H3 nucleosomes suggested that CENP-A nucleosomes might be tetrameric, but new biophysical measurements of reconstituted nucleosomes show that CENP-A confers a reduction in height on octameric CENP-A nucleosomes.

The bacterial transporter XylE is a member of the major facilitator family (MFS). The structure of XylE in its partially occluded outward-facing state was recently solved. Now the same transporter is captured in different conformational states, inward open and partially occluded inward open, thus providing insights into the transport cycle of MFS transporters.