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Biomedical subjects

Syuzo Kaneko

Publications and source records attributed to Syuzo Kaneko.

5 recordsLinked to original sources
PubMed2006

Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.

Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3'-end. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known. Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 A resolution, complexed with zinc ions and a sulphate that might mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1) at 2.5 A resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-beta-lactamase domain and a novel beta-CASP (named for metallo-beta-lactamase, CPSF, Artemis, Snm1, Pso2) domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.

Binding Sites↗
PubMed2005

The mammalian RNA polymerase II C-terminal domain interacts with RNA to suppress transcription-coupled 3' end formation.

RNA polymerase II plays a critical role not only in transcription of mRNA precursors but also in their subsequent processing. This later function is mediated primarily by the C-terminal domain (CTD) of the enzyme's largest subunit, a unique, repetitive structure conserved throughout eukaryotes and known to interact with a number of different proteins during the transcription cycle. Here, we show that the mammalian CTD also interacts with RNA in a sequence-specific manner. We use a variety of RNA binding assays, including SELEX, to characterize the interaction in vitro and a modified chromatin immunoprecipitation (ChIP) assay to provide evidence that it also occurs in vivo. Transfection assays with the CTD binding consensus situated downstream of a polyadenylation signal indicate that the sequence can suppress mRNA 3' end formation and transcription termination, and in vitro assays indicate that the inhibition of processing is CTD dependent. Our results provide an unexpected function for CTD in modulating gene expression.

3' Untranslated Regions↗
PubMed2005

BRCA1/BARD1 inhibition of mRNA 3' processing involves targeted degradation of RNA polymerase II.

Mammalian cells exhibit a complex response to DNA damage. The tumor suppressor BRCA1 and associated protein BARD1 are thought to play an important role in this response, and our previous work demonstrated that this includes transient inhibition of the pre-mRNA 3' processing machinery. Here we provide evidence that this inhibition involves proteasomal degradation of a component necessary for processing, RNA polymerase II (RNAP II). We further show that RNAP IIO, the elongating form of the enzyme, is a specific in vitro target of the BRCA1/BARD1 ubiquitin ligase activity. Significantly, siRNA-mediated knockdown of BRCA1 and BARD1 resulted in stabilization of RNAP II after DNA damage. In addition, inhibition of 3' cleavage induced by DNA damage was reverted in extracts of BRCA1-, BARD1-, or BRCA1/BARD1-depleted cells. We also describe corresponding changes in the nuclear localization and/or accumulation of these factors following DNA damage. Our results support a model in which a BRCA1/BARD1-containing complex functions to initiate degradation of stalled RNAP IIO, inhibiting the coupled transcription-RNA processing machinery and facilitating repair.

3' Flanking Region↗
PubMed2002

Requirements of the RNA polymerase II C-terminal domain for reconstituting pre-mRNA 3' cleavage.

RNA polymerase II (RNAP II) has previously been shown to be required for the pre-mRNA polyadenylation cleavage reaction in vitro. This activity was found to reside solely in the C-terminal domain (CTD) of the enzyme's largest subunit. Using a deletion analysis of glutathione S-transferase-CTD fusion proteins, we searched among the CTD's 52 imperfectly repetitive heptapeptides for the minimal subset that possesses this property. We found that heptads in the vicinity of 30 to 37 contribute modestly more than other sections, but that no specific subsection of the CTD is necessary or sufficient for cleavage. To investigate further the heptad requirements for cleavage, we constructed a series of all-consensus CTDs having 13, 26, 39, and 52 YSPTSPS repeats. We found that the nonconsensus CTD heptads are together responsible for only 20% of the wild-type cleavage activity. Analysis of the all-consensus CTD series revealed that the remaining 80% of the CTD-dependent cleavage activity directly correlates with CTD length, with significant activity requiring approximately 26 or more repeats. These results are consistent with a scaffolding role for the RNAP II CTD in the pre-mRNA cleavage reaction.

3' Flanking Region↗