9 research outputs found
3'-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors
3′-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3′-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3′-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem–loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation—CPSF, CstF, and CF Im—are present in Drosophila nuclear extracts in a stable supercomplex
Comprehensive nucleosome interactome screen establishes fundamental principles of nucleosome binding
Nuclear proteins bind chromatin to execute and regulate genome-templated processes. While studies of individual nucleosome interactions have suggested that an acidic patch on the nucleosome disk may be a common site for recruitment to chromatin, the pervasiveness of acidic patch binding and whether other nucleosome binding hot-spots exist remain unclear. Here, we use nucleosome affinity proteomics with a library of nucleosomes that disrupts all exposed histone surfaces to comprehensively assess how proteins recognize nucleosomes. We find that the acidic patch and two adjacent surfaces are the primary hot-spots for nucleosome disk interactions, whereas nearly half of the nucleosome disk participates only minimally in protein binding. Our screen defines nucleosome surface requirements of nearly 300 nucleosome interacting proteins implicated in diverse nuclear processes including transcription, DNA damage repair, cell cycle regulation and nuclear architecture. Building from our screen, we demonstrate that the Anaphase-Promoting Complex/Cyclosome directly engages the acidic patch, and we elucidate a redundant mechanism of acidic patch binding by nuclear pore protein ELYS. Overall, our interactome screen illuminates a highly competitive nucleosome binding hub and establishes universal principles of nucleosome recognition
Activities of Topoisomerase I in Its Complex with SRSF1
Human DNA topoisomerase I (topo I) catalyzes DNA relaxation
and
phosphorylates SRSF1. Whereas the structure of topo I complexed with
DNA has been resolved, the structure of topo I in the complex with
SRSF1 and structural determinants of topo I activities in this complex
are not known. The main obstacle to resolving the structure is a contribution
of unfolded domains of topo I and SRSF1 in formation of the complex.
To overcome this difficulty, we employed a three-step strategy: identifying
the interaction regions, modeling the complex, and validating the
model with biochemical methods. The binding sites in both topo I and
SRSF1 are localized in the structured regions as well as in the unfolded
domains. One observes cooperation between the binding sites in topo
I but not in SRSF1. Our results indicate two features of the unfolded
RS domain of SRSF1 containing phosphorylated residues that are critical
for the kinase activity of topo I: its spatial arrangement relative
to topo I and the organization of its sequence. The efficiency of
phosphorylation of SRSF1 depends on the length and flexibility of
the spacer between the two RRM domains that uniquely determine an
arrangement of the RS domain relative to topo I. The spacer also influences
inhibition of DNA nicking, a prerequisite for DNA relaxation. To be
phosphorylated, the RS domain has to include a short sequence recognized
by topo I. A lack of this sequence in the mutants of SRSF1 or its
spatial inaccessibility in SRSF9 makes them inadequate as topo I/kinase
substrates