| dc.description.abstracteng | The spliceosome is a highly dynamic, megadalton ribonucleoprotein (RNP) molecular
machine that catalyses the important step of the post-transcriptional processing of eukaryotic
precursor messenger RNA (pre-mRNA), called splicing. During splicing the non-coding
nucleotide intervening regions (introns) of newly transcribed pre-mRNA are excised and the
protein-coding sequences (exons) are ligated via two consecutive transesterification reactions.
The spliceosome is assembled de novo on each pre-mRNA intron by consecutive recruitment
of five RNA-protein complexes (snRNPs) and numerous non-snRNP factors in a dynamic
manner. Initially, the U1 and U2 snRNPs bind to the 5’ splice site and branch point sequence,
respectively, forming the A complex. In the next step, the preformed U4/U6.U5 tri-snRNP
binds and complex B is assembled. Thereafter, complex B undergoes multiple rearrangements
in terms of both its conformation and composition, including the dissociation of U1 and U4,
resulting in the formation of the Bact complex. After the formation of the catalytically active
B* complex, the first step of splicing takes place, generating the cleaved 5' exon and intron-3'
exon lariat intermediates, and creating complex C, which performs the second step of
splicing, resulting in the ligation of the 5' and 3' exons and release of an intron in the form of
a lariat. Various assembly intermediates of the spliceosome have been experimentally defined
and characterized. However, the dynamics of protein exchange, as well as RNA
rearrangements during the spliceosome assembly remain in many aspects unclear.
The spliceosome assembly is highly regulated in multiple ways. Disruption or misregulation
of alternative and constitutive splicing are the cause or severity modulator of many human
diseases, including among others cancer, neurodegenerative and autoimmune diseases,
making the spliceosome a highly attractive drug target. Small molecule inhibitors that block
discrete steps of the extremely dynamic assembly and functional cycle of the spliceosome are
not only of potential therapeutic value, but also highly useful for the detailed investigation of
the structure and function of the spliceosome. However, only a limited number of small
molecule inhibitors that specifically target the pre-mRNA splicing machinery have been
identified at present. Using a robust, rapid and sensitive high throughput in vitro splicing
assay, which monitors the formation of step I spliceosomes (i.e. the spliceosomal C complex)
by measuring the association of a FLAG-tagged version of the DEAD box ATPase Abstrakt,
a chemical library of ~172,000 small molecules was screened for splicing inhibition activity.
All compounds showing at least a 50% decrease in the signal intensity of bound Abstrakt
were subjected to a second test, and reproducible hits were finally tested in an in vitro
splicing assay using 32P-labeled adenovirus-derived MINX-MS2 pre-mRNA as substrate.
Ten compounds were confirmed to be inhibitors of pre-mRNA splicing in vitro, and exhibited
IC50 values ranging from 3 to 50 µM, To determine at which stage they inhibit splicing, I performed a splicing time course and analysed the spliceosomal complexes formed by native
agarose gel electrophoresis. Analysis of splicing complex formation revealed that at least one
compound (hereafter designated as 028), led to an accumulation of A complexes and a
complex denoted as B028 that sediments slightly faster than the pre-catalytic B complex on
glycerol gradients. Gradient centrifugation experiments indicated that U4/U6.U5 tri-snRNP
stability is not affected significantly by 028. To characterize in more detail spliceosomal
complexes formed in the presence of compound 028, I subjected the stalled splicing reactions
to glycerol-gradient centrifugation and purified the complexes in a given peak by MS2-MBP
affinity selection. Initial analysis of the RNA and protein composition of complexes affinity-
purified from the “B-like” peak, suggested that compound 028 stalls splicing at a novel stage
of the spliceosome activation step; i.e., inhibition appears to take place after release of the U4
snRNP, but prior to the release of the LSm and B-specific proteins and before the stable
integration of the Prp19 complex, the recruitment of Bact specific proteins and the
phosphorylation of U2 protein SF3b155. Analysis of the RNA-RNA network within B028 by
RNA structure probing and psoralen crosslinking suggested, but did not definitively prove,
that the catalytically important U6-ISL and U2/U6 helix Ia and Ib likely are formed in the
spliceosomes stalled by compound 028. Purified B028-complexes could be chased into
catalytically active spliceosomes upon addition of microccocal nuclease-treated nuclear
extract, demonstrating that they are functional complexes. We thus performed initial “chase”
experiments with purified B028 complexes to follow the recruitment and release of proteins
during the activation step. Our results suggest that ATP is required for the recruitment of the
Prp19/CDC5L complex proteins and B-specific proteins. Negative stain electron microscopy
of B028-complexes and 3D reconstruction of the B028-particle revealed a unique morphology,
which is different from the pre-catalytic B and activated Bact spliceosomal complexes, but
suggest common morphological features with both B and Bact, consistent with them being
stalled at an intermediate assembly stage. Finally, structure-activity relationship (SAR)
studies, in which modifications of compound 028 were assayed for their effect on pre-mRNA
splicing in vitro, revealed structural determinants that are important for the inhibition activity
of compound 028. Thus, compound 028 allows us to obtain a novel snapshot of the
spliceosome assembly pathway and to perform detailed structural and functional
investigations, to improve our limited understanding of the dynamic rearrangement of
spliceosomal components during spliceosome activation. | de |