A specialised SKI complex assists the cytoplasmic RNA exosome in the absence of direct association with ribosomes.

The Ski2-Ski3-Ski8 (SKI) complex assists the RNA exosome during the 3' to 5' degradation of cytoplasmic transcripts. Previous reports showed that the SKI complex is involved in the 3' to 5' degradation of mRNAs, including 3' untranslated regions (UTRs) and devoid of ribosomes. Paradoxically, we recently showed that the SKI complex directly interacts with ribosomes during the co-translational mRNA decay and that this interaction is necessary for its RNA degradation promoting activity. Here, we characterised a new SKI-associated factor, Ska1, that associates with a subpopulation of the SKI complex. We showed that Ska1 is specifically involved in the degradation of long 3'UTR-containing mRNAs, poorly translated mRNAs as well as other RNA regions not associated with ribosomes, such as cytoplasmic lncRNAs. We further show that the overexpression of SKA1 antagonises the SKI-ribosome association. We propose that the Ska1-SKI complex assists the cytoplasmic exosome in the absence of direct association of the SKI complex with ribosomes.

Rqc1 and Ltn1 Prevent C-terminal Alanine-Threonine Tail (CAT-tail)-induced Protein Aggregation by Efficient Recruitment of Cdc48 on Stalled 60S Subunits.

Protein homeostasis is maintained by quality control mechanisms that detect and eliminate deficient translation products. Cytosolic defective proteins can arise from translation of aberrant mRNAs lacking a termination codon (NonStop) or containing a sequence that blocks translation elongation (No-Go), which results in translational arrest. Stalled ribosomes are dissociated, aberrant mRNAs are degraded by the cytoplasmic exosome, and the nascent peptides remaining in stalled 60S exit tunnels are detected by the ribosome-bound quality control complex (RQC) composed of Ltn1, Rqc1, Rqc2, and Cdc48. Whereas Ltn1 polyubiquitylates these nascent peptides, Rqc2 directs the addition of C-terminal alanine-threonine tails (CAT-tails), and a Cdc48 hexamer is recruited to extract the nascent peptides, which are addressed to the proteasome for degradation. Although the functions of most RQC components have been described, the role of Rqc1 in this quality control process remains undetermined. In this article we show that the absence of Rqc1 or Ltn1 results in the aggregation of aberrant proteins, a phenomenon that requires CAT-tail addition to the nascent peptides by Rqc2. Our results suggest that aberrant CAT-tailed protein aggregation results from a defect in Cdc48 recruitment to stalled 60S particles, a process that requires both Rqc1 and Ltn1. These protein aggregates contain Ltn1-dependent polyubiquitin chains and are degraded by the proteasome. Finally, aggregate characterization by proteomics revealed that they contain specific chaperones including Sis1, Sgt2, Ssa1/2, and Hsp82, suggesting that these protein aggregates may be addressed to aggresome-like structures when the RQC complex fails to deliver aberrant nascent peptides to the proteasome for degradation.

RNA mimicry by the fap7 adenylate kinase in ribosome biogenesis.

During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53-MDM2 pathway. This work presents the functional and structural characterization of the Fap7-Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.

Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7.

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3' hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5'-end with the foot of the 3' hairpin and has thus functional implications.

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits.

During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.