Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm1,2. Hundreds of assembly factors, organized into sequential functional groups3,4, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.

DNA replication in eukaryotes is strictly regulated by several mechanisms. A central step in this replication is the assembly of the heterohexameric minichromosome maintenance (MCM2-7) helicase complex at replication origins during G1 phase as an inactive double hexamer. Here, using cryo-electron microscopy, we report a near-atomic structure of the MCM2-7 double hexamer purified from yeast G1 chromatin. Our structure shows that two single hexamers, arranged in a tilted and twisted fashion through interdigitated amino-terminal domain interactions, form a kinked central channel. Four constricted rings consisting of conserved interior β-hairpins from the two single hexamers create a narrow passageway that tightly fits duplex DNA. This narrow passageway, reinforced by the offset of the two single hexamers at the double hexamer interface, is flanked by two pairs of gate-forming subunits, MCM2 and MCM5. These unusual features of the twisted and tilted single hexamers suggest a concerted mechanism for the melting of origin DNA that requires structural deformation of the intervening DNA.

Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.

Kinetics and cryo-electronmicroscopy data provide insights into GTPase ObgE’s role as a ribosome anti-association factor that is modulated by nutrient availability, coupling growth control to ribosome biosynthesis and protein translation.

Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3'-end for maturation. A crucial step in this process is the recognition of the AAUAAA polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here, we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-Å resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3'-end formation.

No abstract

Autophagic lysosome reformation (ALR) plays an important role in maintaining lysosome homeostasis. During ALR, lysosomes are reformed by recycling lysosomal components from autolysosomes. The most noticeable step of ALR is autolysosome tubulation, but it is currently unknown how the process is regulated. Here, using an approach combining in vivo studies and in vitro reconstitution, we found that the kinesin motor protein KIF5B is required for autolysosome tubulation and that KIF5B drives autolysosome tubulation by pulling on the autolysosomal membrane. Furthermore, we show that KIF5B directly interacts with PtdIns(4,5)P2. Kinesin motors are recruited and clustered on autolysosomes via interaction with PtdIns(4,5)P2 in a clathrin-dependent manner. Finally, we demonstrate that clathrin promotes formation of PtdIns(4,5)P2-enriched microdomains, which are required for clustering of KIF5B. Our study reveals a mechanism by which autolysosome tubulation was generated.

Highlights d An mPSF interaction motif (PIM) in CPSF100 tethers mCF to mPSF in CPSF d The overall conformation of mCF and its location relative to mPSF are highly dynamic d CstF is bound to mPSF through its CstF77 subunit d CPSF160 and WDR33 of mPSF form the core of the pre-mRNA 3 0 -end-processing machinery