Catalytic activation of human spliceosomes by RNA helicases

RNA helicases are molecular motors that convert the energy stored in ATP into various rearrangements of RNA and RNA-protein complexes, keeping the RNA metabolism dynamic and tightly regulated. Spliceosomes, which are the largest and most complex nanomachines that process genetic information, require numerous helicases to regulate their function. Using cryo-EM of stalled spliceosome and biochemical methods, we found that human spliceosomes need to be remodelled by two helicases – PRP2 and Aquarius, to kick-start the splicing reaction. 

Identification of a spliceosome stalled halfway through the catalytic activation (Schmitzova et al., Nature, 2023).

Mechanism of the helicase PRP2 in splicing 

The DEAH helicases are special as they can grasp an RNA strand and translocate along nucleotide by nucleotide while hydrolyzing ATP molecules. Four DEAH helicases are essential for splicing, although their mechanism of action in splicing remained unclear, as they were observed at fixed positions on the periphery of the spliceosomes. The BAQR spliceosome, found halfway through the catalytic activation, has finally shown how a DEAH helicase, PRP2, translocates about 19 nucleotides from the periphery towards the core of the spliceosome while remodelling the RNA-protein contacts, changing conformations, dissociating and recruiting proteins. 

The helicase PRP2 translocates 19 nucleotides at the transition from Bact to BAQR (Schmitzova et al., Nature, 2023).

General mechanism of intron recognition during spliceosome assembly

The structure of a cross-exon spliceosome stalled by SSA captures the intron recognition by U2 snRNP.
This indicates that early prespliceosomes (E complexes) select introns through a toehold-mediated strand displacement mechanism. Strand displacement is primarily known from the DNA field, on processes like the DNA strand exchange, and in applied DNA nanotechnologies. 

The structure of the exon-definition complex core stalled with spliceostatin (above) and the mechanism of intron recognition by strand displacement (below; Cretu et al., Nat. Comm., 2021).

General mechanism of splicing modulation by SF3B ligands

SF3B ligands with antitumor properties act as competitive antagonists of branch sites (see SF3B-PB, SF3B-SSA, SF3B-SUD). Our structures explain why the inhibition depends on the sequence motifs of the intron, as such that some branch sites can be inactivated more readily than others. The modulation manifests as differential inhibitions (some branch sites will be used while some others not), resulting in a change in AS pattern (Cretu et al., Mol. Cell, 2018; Nat Commun 2021).

Splicing modulators that bind SF3B1 act as competitive antagonists of the branch site, in a manner dependent on the intron’s sequence (Cretu et al., Mol. Cell, 2018; Nat. Comm., 2021)

Mechanism of covalent coupling of spliceostatin and sudemycine to a zinc finger of SF3B

Our crystal structures have revealed that the long-studied compounds from the spliceostatin family and their relatives, sudemycins, bind prespliceosomes by covalent coupling to a zinc finger of the PHF5A protein. The central conjugated diene of the compounds acts as a spacer for the warhead epoxy group that reacts with a thiol of the cysteine. 

Spliceostatin and sudemycins bind spliceosomes by covalent coupling to a zing finger from the SF3B subunit PHF5A.

The NineTeen Complex (NTC) promotes ubiquitination in DNA repair

As part of the heteromeric NineTeen Complex (NTC), Prp19 is a homotetramer complex that promotes ubiquitination during the formation of the spliceosomal tri-snRNP particle and DNA damage response. We showed by X-ray crystallography and functional analyses in vitro and in vivo that Prp19 is an autoinhibited E3 ubiquitin ligase that becomes active only in stable association with three other NTC components that exert specific effects on Prp19's conformation. (De Moura et al., Mol. Cell, 2018).

PRP19 is an autoinhibited E3 ligase activated by stepwise assembly of three splicing factors, together forming the NTC’s core

The Intron-binding complex (IBC) enables recruitment of the splicing-essential helicase Aquarius (AQR, IBP160) to the human spliceosomes

The human helicase Aquarius induces an additional ATP-dependent remodeling of the spliceosome. We determined the structure of Aquarius and showed that this helicase is recruited to the spliceosome as part of the pentameric intron binding complex (IBC).

Crystal structure of the RNA helicase Aquarius

Crystal structure of a DNA catalyst

Although DNA is known mainly for its capacity to encode genetic information, DNA molecules that catalyze various chemical reactions have been found in vitro. Two decades after this discovery, we have determined the first crystal structure of a DNA enzyme. The surprisingly complex fold that these molecules adopt raises questions about the potential structural importance of DNA in the cell (Ponce-Salvatierra et al., Nature 2016).

Crystal structure of the deoxyribozyme 9DB1 in complex with the RNA product