A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3

Abstract

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene. Transcription terminates via the Nrd1-Nab3-Sen1 complex and is degraded by the nuclear exosome. Using a sensitive terminator read-through assay, we identified trans-acting Terminator Override (TOV) genes that operate this terminator. Four genes were identified: the RNA polymerase II phosphatase SSU72, the RNA polymerase II binding protein PCF11, the TRAMP subunit TRF4 and the hnRNP-like, NAB3. The TOV phenotype can be explained by the loss of function of these gene products as described in models in which termination and RNA degradation are coupled to the phosphorylation state of RNA polymerase II's repeat domain. The most interesting mutations were those found in NAB3, which led to the finding that the removal of merely three carboxy-terminal amino acids compromised Nab3's function. This region of previously unknown function is distant from the protein's well-known RNA binding and Nrd1 binding domains. Structural homology modeling suggests this Nab3 ‘tail’ forms an α-helical multimerization domain that helps assemble it onto an RNA substrate.

A Network of Interdependent Molecular Interactions Describes a Higher Order Nrd1-Nab3 Complex Involved in Yeast Transcription Termination

“…The lithium acetate method of transformation was used throughout. Diploid strains were generated by mating the nab3C⌬19-containing strain 1A1F (15,16) or its otherwise isogenic wild type strain BY4742 to the nrd1⌬151-214-containing strain, YSB2078 (13). These diploids, DY2098 and DY2097, respectively, were then transformed with pRS316-NRD1 and sporulated.…”

“…Intriguing-A spontaneous mutation that removes 19 residues from the S. cerevisiae Nab3 C terminus compromises termination and self-association of the protein (15,16). This region contains a stretch of 16 glutamines adjacent to 18 amino acids with homology to a human hnRNP ␣-helix responsible for hnRNP-C tetramerization (Fig.…”

“…1B). Although cells grew slowly when the Nab3 hnRNP-C homology region was removed, they remain viable (15,16). Prior deletion analysis indicated that there was an essential region encompassed in the C-terminal 238 amino acids (1).…”

“…Using a genetic screen, we have recently discerned a function for the very C-terminal tail of Nab3 (Fig. 1, A (circled number 4) and B) (15,16). It contains a stretch of 16 glutamines adjacent to a short peptide that shares structural homology with an ␣-helix of the human hnRNP-C protein (15,16).…”

“…1, A (circled number 4) and B) (15,16). It contains a stretch of 16 glutamines adjacent to a short peptide that shares structural homology with an ␣-helix of the human hnRNP-C protein (15,16). Both elements of this "tail" region are involved in self-association, similar to the tetramerization helix of human hnRNP-C (17).…”