Evolutionary and functional insights into the Ski2-like helicase family in Archaea: a comparison of Thermococcales ASH-Ski2 and Hel308 activities.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The Ski2-like proteins are primordial helicases that play an active role in eukaryotic RNA homeostasis pathways, with multiple homologs having specialized functions. The significance of the expansion and diversity of Ski2-like proteins in Archaea, the third domain of life, has not yet been established.

The DEAD-box protein Dbp6 is an ATPase and RNA annealase interacting with the peptidyl transferase center (PTC) of the ribosome.

Ribosomes are ribozymes, hence correct folding of the rRNAs during ribosome biogenesis is crucial to ensure catalytic activity. RNA helicases, which can modulate RNA-RNA and RNA/protein interactions, are proposed to participate in rRNA tridimensional folding. Here, we analyze the biochemical properties of Dbp6, a DEAD-box RNA helicase required for the conversion of the initial 90S pre-ribosomal particle into the first pre-60S particle.

Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast.

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA.

Phylogenetic Diversity of Lhr Proteins and Biochemical Activities of the Thermococcales aLhr2 DNA/RNA Helicase.

Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized.

The G-patch activators Pfa1 and PINX1 exhibit different modes of interaction with the Prp43 RNA helicase.

Prp43 is a DEAH-box RNA helicase involved in both splicing and ribosome biogenesis. Its activities are directly stimulated by several co-activators that share a G-patch domain. The substrates of Prp43, its mechanism of action and the modes of interaction with and activation by G-patch proteins have been only partially characterized.

The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor.

The early steps of the production of the large ribosomal subunit are probably the least understood stages of eukaryotic ribosome biogenesis. The first specific precursor to the yeast large ribosomal subunit, the first pre-60S particle, contains 30 assembly factors (AFs), including 8 RNA helicases. These helicases, presumed to drive conformational rearrangements, usually lack substrate specificity in vitro.

Functional link between DEAH/RHA helicase Prp43 activation and ATP base binding.

The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism. In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family.

The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis.

We provide evidence that a central player in ribosome synthesis, the ribonucleic acid helicase Prp43p, can be activated by yeast Gno1p and its human ortholog, the telomerase inhibitor PINX1. Gno1p and PINX1 expressed in yeast interact with Prp43p and the integrity of their G-patch domain is required for this interaction. Moreover, PINX1 interacts with human PRP43 (DHX15) in HeLa cells.

Prp43p contains a processive helicase structural architecture with a specific regulatory domain.

The DEAH/RNA helicase A (RHA) helicase family comprises proteins involved in splicing, ribosome biogenesis and transcription regulation. We report the structure of yeast Prp43p, a DEAH/RHA helicase remarkable in that it functions in both splicing and ribosome biogenesis. Prp43p displays a novel structural architecture with an unforeseen homology with the Ski2-like Hel308 DNA helicase.

The ATPase and helicase activities of Prp43p are stimulated by the G-patch protein Pfa1p during yeast ribosome biogenesis.

Prp43p is a RNA helicase required for pre-mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G-patch protein Pfa1p, a component of pre-40S pre-ribosomal particles, directly interacts with Prp43p.

Analysis of the binding of the N-terminal conserved domain of yeast Cbf5p to a box H/ACA snoRNA.

During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p.

Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs.

Box C/D and box H/ACA small ribonucleoprotein particles (sRNPs) are found from archaea to humans, and some of these play key roles during the biogenesis of ribosomes or components of the splicing apparatus. The protein composition of the core of both types of particles is well established and the assembly pathway of box C/D sRNPs has been extensively investigated both in archaeal and eukaryotic systems. In contrast, knowledge concerning the mode of assembly and final structure of box H/ACA sRNPs is much more limited.