Summary
Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3ʹ ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3ʹ end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit “CPF
core
” complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPF
core
. Together, our data suggest that the active mRNA 3ʹ end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.
Investigations into
the chemical origin of life have recently benefitted
from a holistic approach in which possible atmospheric, organic, and
inorganic systems chemistries are taken into consideration. In this
way, we now report that a selective phosphate activating agent, namely
methyl isocyanide, could plausibly have been produced from simple
prebiotic feedstocks. We show that methyl isocyanide drives the conversion
of nucleoside monophosphates to phosphorimidazolides under potentially
prebiotic conditions and in excellent yields for the first time. Importantly,
this chemistry allows for repeated reactivation cycles, a property
long sought in nonenzymatic oligomerization studies. Further, as the
isocyanide is released upon irradiation, the possibility of spatially
and temporally controlled activation chemistry is thus raised.
It all clicks into place: A potent telomere‐targeting small molecule has been identified by using the copper‐free 1,3‐dipolar cycloaddition of a series of alkyne and azide building blocks catalyzed by a non‐Watson–Crick DNA secondary structure (see picture). This method rapidly identifies, otherwise unanticipated, potent small‐molecule probes to selectively target a given RNA or DNA.
Replication
of nucleic acids in the absence of genetically encoded
enzymes represents a critical process for the emergence of cellular
life. Repeated separation of complementary RNA strands is required
to achieve multiple cycles of chemical replication, yet thermal denaturation
under plausible prebiotic conditions is impaired by the high temperatures
required to separate long RNA strands and by concurrent degradation
pathways, the latter accelerated by divalent metal ions. Here we show
how the melting temperature of oligoribonucleotide duplexes can be
tuned by changes in pH, enabling the separation of RNA strands at
moderate temperatures. At the same time, the risk of phosphodiester
bond cleavage is reduced under the acid denaturation conditions herein
described, both in the presence and in the absence of divalent metal
ions. Through a combination of ultraviolet and circular dichroism
thermal studies and gel electrophoresis, we demonstrate the relevance
of geological pH oscillations in the context of the RNA strand separation
problem. Our results reveal new insights in the field of prebiotic
chemistry, supporting plausible geochemical scenarios in which non-enzymatic
RNA replication might have taken place.
The complexity of the simplest conceivable cell suggests that the chemistry of prebiotic mixtures needs to be explored to understand the intricate network of prebiotic reactions that led to the emergence of life.
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