Interaction of yeast RNA-binding proteins Nrd1 and Nab3 with RNA polymerase II terminator elements

Abstract

Yeast RNA-binding proteins Nrd1 and Nab3 direct transcription termination of sn/snoRNA transcripts, some mRNA transcripts, and a class of intergenic and anti-sense transcripts. Recognition of Nrd1- and Nab3-binding sites is a critical first step in the termination and subsequent processing or degradation of these transcripts. In this article, we describe the purification and characterization of an Nrd1-Nab3 heterodimer. This Nrd1-Nab3 complex binds specifically to RNA sequences derived from a snoRNA terminator. The relative binding to mutant terminators correlates with the in vivo termination efficiency of these mutations, indicating that the primary specificity determinant in nonpoly(A) termination is Nrd1-Nab3 binding. In addition, several snoRNA terminators contain multiple Nrd1- and Nab3-binding sites and we show that multiple heterodimers bind cooperatively to one of these terminators in vitro.

FIGURE 1.

Expression and purification of Nrd1–Nab3 heterodimer. (A) Schematic of the regions of Nrd1 and Nab3 included in the expression vector. Nrd1 was His₉ tagged at the N terminus and Nab3 was MBP tagged at the N terminus. (B) SDS-PAGE of purified protein.

FIGURE 2.

Binding affinity and specificity of Nrd1–Nab3 heterodimer. (A) Sequence of U6R* RNA oligos. (B) Binding curves of U6R* RNA oligos to Nrd1–Nab3. (C) Sequence of snR13 RNA oligos. (D) Binding curves of WT and single mutant snR13 RNA oligos to Nrd1–Nab3. (E) Binding curves of WT and double mutant snR13 RNA oligos to Nrd1–Nab3. Nrd1 (NR) and Nab3 (NA) motifs are underlined in red. Single base changes in the binding motifs are highlighted. Filter binding data were fit to a four parameter logistic equation using GraphPad Prism software.

FIGURE 3.

Termination efficiency of snR13 WT and double mutant variants. (A) Schematic of GFP readthrough vectors. (Middle) The SNR13 gene in its genomic locus. (Top) A plasmid containing the SNR13 promoter, gene, and 108 base pairs (bp) of downstream sequence. (Bottom) A plasmid containing 35 bp of snR13 downstream sequence driven by the ADH promoter. Vertical dashed lines indicate regions of identical sequence. Wavy lines indicate the vector. (B) Northern blot of total RNA from yeast harboring the GFP readthrough plasmids. GFP signal was corrected with loading control to SCR1 and is an average of two experiments. Lanes 2 and 7 contain vectors with no snR13 downstream sequences and represent maximum transcription readthrough. These maxima were set to 1 and all other lanes were expressed as a fraction of that maximum.

FIGURE 4.

Nrd1–Nab3 terminators can bind multiple heterodimers. (A) Gel shift of U6R* RNA oligos and Nrd1–Nab3 protein. (B) Gel shift of snR13 RNA oligos and Nrd1–Nab3 protein. (C) Gel shift of panel of snR13 RNA oligos at 5 μM Nrd1–Nab3 protein. Wedge represents increasing concentration of Nrd1–Nab3 heterodimer: 0, 0.156, 0.312, 0.625, 1.25, 2.5, and 5 μM.

FIGURE 5.

Footprint of Nrd1–Nab3 on RNA. (A) RNase A footprinting of U6R* RNA oligos and Nrd1–Nab3. RNase A cuts after cytosine and uridine residues in single-stranded RNA. The WT U6R* sequence is indicated at the bottom with the NA mutant RNA sequence change underneath. (B) RNase T1 footprinting of snR13 RNA oligos and Nrd1–Nab3. RNase T1 cuts after guanosine residues in single-stranded RNA. The WT snR13 sequence is indicated at the bottom with the NR1 and NA1 mutant RNA sequence changes underneath. The “C” indicates a control lane containing no RNase. Wedge represents increasing concentration of Nrd1–Nab3 heterodimer: 0, 0.625, 1.25, and 2.5 μM. Mutations are indicated by an asterisk. Colored squares represent bases in an Nrd1 or a Nab3 motif and colored circles represent additional bases along the RNA. Locations on the RNA oligo and corresponding position on the gel are denoted by the squares and circles.

FIGURE 6.

Sedimentation velocity experiments on Nrd1–Nab3 and its complexes with RNA. c(s) distributions based on sedimentation velocity data collected at 55 krpm, 280 nm, and 4.0°C are shown. (A) The distribution for Nrd1–Nab3 at a loading concentration of 10 μM. (B) The distributions for a 1:1.05 Nrd1–Nab3 to snR13 11-nt RNA mixture at 6.7 μM. Data for the complex at 6.7 μM can be modeled in terms of a single species with an s_20,w of 4.9 S. The c(s) distributions for Nrd1–Nab3 and snR13 35 nt RNA at nominal loading concentrations of (C) 1:2 (2.75 μM Nrd1–Nab3), (D) 1:1 (5.5 μM Nrd1–Nab3), (E) 1.3:1 (5.5 μM Nrd1–Nab3), (F) 2.2:1 (7.1 μM Nrd1–Nab3), and (G) 4.4:1 (7.4 μM Nrd1–Nab3) protein:RNA. Note the progressive transition from a 1:1 Nrd1–Nab3 to RNA 5.6 S complex, to a 2:1 Nrd1–Nab3 to RNA 8.1 S complex. Excess RNA for the 1:2, 1:1, and 1.3:1 mixtures is noted at sedimentation values <4. The gray lines highlight the sedimentation coefficients of the free Nrd1–Nab3, the 1:1 complex with snR13 35-nt RNA, and the 2:1 complex with the same RNA.

FIGURE 7.

Model of Nrd1–Nab3 heterodimer binding to Nrd1–Nab3 RNA terminators. (A) The U6R* RNA contains one Nab3 (NA) and one Nrd1 (NR) site and allows for the binding of one Nrd1–Nab3 heterodimer. (B) The snR13 11-nt RNA also binds one Nrd1–Nab3 heterodimer. (C) The snR13 35-nt RNA contains four potential binding sites. The first heterodimer may bind to the central NA1–NR2 site, allowing for the binding of a second heterodimer to the NR1 site or to the NA2 site (not shown). (D) The unoccupied Nab3 side of the heterodimer would then be free to bind the NA2 site of another RNA, thus seeding the formation of larger protein–RNA complexes. (E) Model for Nrd1–Nab3 binding to multiple sites within an mRNA and subsequent interaction of numerous Nrd1 CIDs with multiple CTD repeats.