Nab3 Facilitates the Function of the TRAMP Complex in RNA Processing via Recruitment of Rrp6 Independent of Nrd1

Non-coding RNAs (ncRNAs) play critical roles in gene regulation. In eukaryotic cells, ncRNAs are processed and/or degraded by the nuclear exosome, a ribonuclease complex containing catalytic subunits Dis3 and Rrp6. The TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex is a critical exosome cofactor in budding yeast that stimulates the exosome to process/degrade ncRNAs and human TRAMP components have recently been identified. Importantly, mutations in exosome and exosome cofactor genes cause neurodegenerative disease. How the TRAMP complex interacts with other exosome cofactors to orchestrate regulation of the exosome is an open question. To identify novel interactions of the TRAMP exosome cofactor, we performed a high copy suppressor screen of a thermosensitive air1/2 TRAMP mutant. Here, we report that the Nab3 RNA-binding protein of the Nrd1-Nab3-Sen1 (NNS) complex is a potent suppressor of TRAMP mutants. Unlike Nab3, Nrd1 and Sen1 do not suppress TRAMP mutants and Nrd1 binding is not required for Nab3-mediated suppression of TRAMP suggesting an independent role for Nab3. Critically, Nab3 decreases ncRNA levels in TRAMP mutants, Nab3-mediated suppression of air1/2 cells requires the nuclear exosome component, Rrp6, and Nab3 directly binds Rrp6. We extend this analysis to identify a human RNA binding protein, RALY, which shares identity with Nab3 and can suppress TRAMP mutants. These results suggest that Nab3 facilitates TRAMP function by recruiting Rrp6 to ncRNAs for processing/degradation independent of Nrd1. The data raise the intriguing possibility that Nab3 and Nrd1 can function independently to recruit Rrp6 to ncRNA targets, providing combinatorial flexibility in RNA processing.     

Interaction of the RNA-binding domain of the hnRNP C proteins with RNA.

The hnRNP C proteins are among the most abundant and avid pre-mRNA-binding proteins and they contain a consensus sequence RNA-binding domain (RBD) that is found in a large number of RNA-binding proteins. The interaction of the RBD of the hnRNP C proteins with an RNA oligonucleotide [r(U)8] was monitored by nuclear magnetic resonance (NMR). 15N and 13C/15N-labelled hnRNP C protein RBD was mixed with r(U)8 and one- and two-dimensional (1D and 2D) NMR spectra were recorded in a titration experiment. NMR studies of the uncomplexed 93 amino acid hnRNP C RBD (Wittekind et al., 1992) have shown that it has a compact folded structure (beta alpha beta beta alpha beta), which is typical for the RBD of this family of proteins and which is comprised of a four-stranded antiparallel beta-sheet, two alpha-helices and relatively unstructured amino- and carboxy-terminal regions. Sequential assignments of the polypeptide main-chain atoms of the hnRNP C RBD-r(U)8 complex revealed that these typical structural features are maintained in the complex, but significant perturbations of the chemical shifts of amide group atoms occur in a large number of residues. Most of these residues are in the beta-sheet region and especially in the terminal regions of the RBD. In contrast; chemical shifts of the residues of the well conserved alpha-helices, with the exception of Lys30, are not significantly perturbed. These observations localize the candidate residues of the RBD that are involved in the interaction with the RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

ELA1 and CUL3 are required along with ELC1 for RNA polymerase II polyubiquitylation and degradation in DNA-damaged yeast cells

Treatment of yeast and human cells with DNA-damaging agents elicits lysine 48-linked polyubiquitylation of Rpb1, the largest subunit of RNA polymerase II (Pol II), which targets Pol II for proteasomal degradation. However, the ubiquitin ligase (E3) responsible for Pol II polyubiquitylation has not been identified in humans or the yeast Saccharomyces cerevisiae. Here we show that elongin A (Ela1) and cullin 3 (Cul3) are required for Pol II polyubiquitylation and degradation in yeast cells, and on the basis of these and other observations, we propose that an E3 comprised of elongin C (Elc1), Ela1, Cul3, and the RING finger protein Roc1 (Rbx1) mediates this process in yeast cells. This study provides, in addition to the identification of the E3 required for Pol II polyubiquitylation and degradation in yeast cells, the first evidence for a specific function in yeast for a member of the elongin C/BC-box protein/cullin family of ligases. Also, these observations raise the distinct possibility that the elongin C-containing ubiquitin ligase, the von Hippel-Lindau tumor suppressor complex, promotes Pol II polyubiquitylation and degradation in human cells.