The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome
- Yale University School of Medicine, United States
- National Institute of Environmental Health Sciences, National Institutes of Health, United States
Figures
Figure 1 with 3 supplements
The ANE syndrome mutation confers a growth defect in yeast.
(A) The leucine that is mutated in ANE syndrome is highly conserved. Top: Diagram of the domain structure for human RBM28 and its yeast ortholog, Nop4. The boxes represent RNA Recognition Motifs …
Figure 1—figure supplement 1
Multiple sequence alignment of RRM3 from RBM28.
The amino acids corresponding to RRM3 from human RBM28, yeast Nop4 and RBM28 from M. mulatta, M. musculus, X. tropicalis and D. rerio were aligned. The conserved structural elements of a canonical …
Figure 1—figure supplement 2
RBM28 complements the growth defect in yeast observed upon depletion of its essential ortholog, Nop4.
(A) Nop4 and RBM28 are expressed from the yeast expression vector p414GPD-3xFLAG-GW. Expression of Nop4 (78 kDa) or RBM28 (86 kDa) from p414GPD-3xFLAG-GW was analyzed by western blot using a …
Figure 1—figure supplement 3
Amino acid sequence alignment of human RBM28 and its yeast ortholog Nop4.
The complete amino acid alignment of human RBM28 protein and its yeast ortholog, Nop4, was created using ClustalX. The conserved leucine that is mutated to proline in ANE syndrome, L351 in RBM28 and …
Figure 2
The ANE syndrome mutation disrupts pre-rRNA processing in yeast.
(A) Simplified diagram depicting the pre-rRNA processing steps in yeast. The pre-rRNA is transcribed as a 35S polycistronic precursor. The external transcribed spacers (5´ and 3´ ETS) and the …
-
Figure 2—source data 1
Quantitation and statistical analyses for Figure 2C.
- https://doi.org/10.7554/eLife.16381.008
Figure 3
The ANE syndrome mutation in Nop4 disrupts protein-protein interactions.
(A) Schematic of Y2H analysis. Nop4 WT or Nop4 L306P were cloned into the prey vector (pACT2) while five Nop4 interacting proteins (Noc2, Mak5, Nop4, Nsa2 and Dbp10) were cloned into the bait vector …
-
Figure 3—source data 1
Quantitation and statistical analyses for Figure 3E.
- https://doi.org/10.7554/eLife.16381.010
Figure 4
RRM3 and RRM4 of Nop4 mediate protein-protein interactions.
(A) Schematic representation of Nop4 RRM domains and the N- and C-terminal fragments containing RRMs 1 and 2 (RRM 1–2; residues 1–250) or 3 and 4 (RRM 3–4; residues 252–685), respectively. (B) Nop4 …
Figure 5 with 1 supplement
RRM3 and RRM4 of Nop4 are necessary and sufficient to complement the growth defect due to Nop4 depletion.
(A) Nop4 WT and the Nop4 fragments are differentially expressed from the yeast expression vector p414GPD-3xFLAG-GW. Total protein was extracted from YPH499 GAL::3xHA-NOP4 yeast transformed with …
Figure 5—figure supplement 1
Nop4 RRM 1–2 fails to complement even when targeted to the nucleus.
(A) Expression of the Nop4 fragments from the yeast two-hybrid vector pACT2 ensures nuclear targeting. Yeast expressing 3xHA-Nop4 WT, 3xHA-Nop4 RRM 1–2 or 3xHA-Nop4 RRM 3–4 from the yeast two-hybrid …
Figure 6
Nop4 RRM 3–4 is necessary and sufficient to complement the pre-rRNA processing defect after Nop4 depletion.
(A) Top panel: Total RNA extracted from yeast expressing the indicated Nop4 fragment after depletion of endogenous Nop4 for 24 hr at 30°C or for 48 hr at 23°C was visualized by ethidium bromide …
-
Figure 6—source data 1
Quantitation and statistical analyses for Figure 6B.
- https://doi.org/10.7554/eLife.16381.016
Figure 7
The ANE syndrome mutation, L351P, in human RBM28 disrupts RRM3 domain structure.
(A) Circular dichroism spectra of WT human RBM28 RRM3 (black) and L351P mutant (red). Four technical replicates were performed. (B) 15N-HSQC spectra of WT hRBM28 RRM3 (amino acids 330–419) (black) …
Author response image 1
The ANE syndrome mutation (L306P) disrupts 25S production in yeast.
(A) Left panel: longer exposure of the ethidium bromide stained gel in Figure 2B. Total RNA was extracted from yeast expressing no Nop4 (EV), Nop4 WT or Nop4 L306P after depletion of endogenous Nop4 …
Tables
Table 1
Nop4 interacts with 23 large subunit assembly factors with high confidence. The Nop4 interacting proteins were identified by yeast two-hybrid and were assigned a confidence score in (McCann et al., …
| Nop4 Interacting Partner | Confidence Score (from McCann et al., 2015) |
|---|---|
| Nop4 | 92% |
| Loc1 | 92% |
| Ebp2 | 85% |
| Nop12 | 85% |
| Nsa2 | 85% |
| Mak5 | 84% |
| Cgr1 | 70% |
| Cic1 | 70% |
| Has1 | 70% |
| Noc2 | 70% |
| Nop13 | 70% |
| Nsr1 | 70% |
| Rrp12 | 70% |
| Rrp14 | 70% |
| Mak21 | 68% |
| Dbp10 | 63% |
| Drs1 | 63% |
| Nop16 | 63% |
| Nug1 | 63% |
| Prp43 | 63% |
| Spb4 | 63% |
| Tma16 | 63% |
| Nog1 | 53% |
Additional files
-
Supplementary file 1
Western blot quantitation source data.
- https://doi.org/10.7554/eLife.16381.018
