Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3′-end processing.

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.

The Fanconi Anemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress 1,2. Genetic inactivation of this pathway impairs development, prevents blood production and promotes cancer 1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCI-FANCD2 4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase 6,7. Monoubiquitinated FANCD2 then recruits enzymes to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used electron cryo-microscopy (cryoEM) and mass spectrometry to determine its structure. The FA core complex is comprised of two central dimers of the FANCB and FAAP100 subunits, flanked by two copies of the RING finger protein, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Hunter syndrome is a rare but devastating childhood disease caused by mutations in the IDS gene encoding iduronate-2-sulfatase, a crucial enzyme in the lysosomal degradation pathway of dermatan sulfate and heparan sulfate. These complex glycosaminoglycans have important roles in cell adhesion, growth, proliferation and repair, and their degradation and recycling in the lysosome is essential for cellular maintenance. A variety of disease-causing mutations have been identified throughout the IDS gene. However, understanding the molecular basis of the disease has been impaired by the lack of structural data. Here, we present the crystal structure of human IDS with a covalently bound sulfate ion in the active site. This structure provides essential insight into multiple mechanisms by which pathogenic mutations interfere with enzyme function, and a compelling explanation for severe Hunter syndrome phenotypes. Understanding the structural consequences of disease-associated mutations will facilitate the identification of patients that may benefit from specific tailored therapies.