Figure 1.
The Expression of Rrp6 and Rrp47 is mutually dependent.
Isogenic wild-type and rrp6∆ or rrp47∆ strains were grown in selective minimal medium (SD) or in nonselective rich medium (YPD) and extracts were prepared under alkaline denaturing conditions. Extracts were resolved by SDS-PAGE and western blots were incubated with PAP antibody (Panels A and B) to detect fusion proteins, or with an Rrp6-specific antibody (Panel C). Blots were also incubated with an antibody specific to detect Pgk1, which serves as a loading control. (A) Western analysis of Rrp47-zz in isogenic wild-type RRP6 and rrp6∆ strains. (B) Western analysis of Rrp6-TAP in isogenic wild-type RRP47 and rrp47∆ strains. (C) Western analysis of non-tagged Rrp6 in isogenic wild-type RRP47 and rrp47∆ strains. Relative expression levels of Rrp6 or Rrp47, indicated as percentages under each panel, are normalised for Pgk1 expression levels and standardised to the amount of protein in the wild-type strain grown in YPD. Values are the mean of at least 4 independent experiments.
Figure 2.
Rrp6 protein stability is decreased in the rrp47∆ mutant.
Isogenic wild-type and rrp47∆ strains were harvested during growth in selective minimal medium (SD) or rich medium (YPD) and at time-points after addition of the translation inhibitor cycloheximide (CHX), as indicated. Extracts were prepared under denaturing conditions and identical western blots were incubated with antiserum specific to Rrp6 and the loading control Pgk1. (A) Translational shut-off experiment in YPD medium. (B) Translational shut-off experiment in SD medium. (C) Quantitative analysis of the amount of Rrp6 in extracts from wild-type and rrp47∆ strains before addition of cycloheximide (“0” lanes) and 60 minutes after treatment (“60” lanes). The relative amount of Rrp6, normalised to Pgk1 expression levels and standardised to the level observed in the wild-type strain during growth in YPD medium (average of 2 experiments), is given below each lane.
Figure 3.
RRP6 mRNA levels are decreased in the rrp47∆ mutant.
Relative expression levels of RRP6 mRNA in wild-type and rrp47∆ mutants during growth in either selective minimal medium (SD) or rich medium (YPD). Expression levels, indicated as percentages, are standardised to the amount in wild-type cells grown in YPD medium. RRP6 mRNA levels were determined by qRT-PCR and normalised to the SCR1 RNA. Expression levels of non-tagged Rrp6 protein, determined as in Figure 1, are shown for comparison. Error bars indicate the positive range of the standard error of the mean for each set of values.
Figure 4.
Rrp6 can be overexpressed in wild-type and rrp47∆ strains.
Western analyses of Rrp6 in isogenic wild-type and rrp47∆ strains that are transformed with RRP6 expression constructs. Strains were transformed with either the 2 micron (2μ) vector pRS426 (lane 1), the centromeric (cen) plasmid pRS416 (lane 2), an RRP6 genomic clone in pRS416 (lane 3) or in pRS426 (lane 4), as well as constructs expressing an N-terminal zz fusion of Rrp6 from the RRP4 promoter in pRS416 (lane 5) or pRS426 (lane 6). Identical blots were analysed for Rrp6 levels using an Rrp6-specific antiserum, followed by analysis of the loading control Pgk1.
Figure 5.
Rrp6 overexpression suppresses RNA phenotypes in rrp47∆ mutants.
Northern analyses of total cellular RNA isolated from a wild-type strain, an isogenic rrp47∆ mutant and from rrp47∆ mutants expressing exogenous Rrp6 or Rrp47 from either centromeric (cen) plasmids or 2 micron-based (2μ) constructs. RNA was resolved through 8% denaturing acrylamide gels, transferred to nylon membranes and hybridised with probes complementary to specific RNAs, as follows: (A) U14; (B) snR38; (C) snR13; (D) SCR1; (E) U6; (F) U3; (G) 5.8S; (H) NEL025c; (I) SCR1. Blots shown in A-G and H-I are from distinct gels. Dispersed bands labelled I-pA and II-pA in panels A-C represent snoRNAs that are polyadenylated after termination at sites I and II, respectively. The bands labelled snR13* and U3* are 5’ truncated forms of snR13 and U3. (J) Quantification of signals for the 3’ extended forms of U14, snR38, snR13, U6 and 5.8S, the truncated U3 RNA and the NEL025c mRNA are shown for each strain. Average values of two data sets are normalised to SCR1 loading controls and expressed relative to the level of the RNA observed in the wild-type strain.
Figure 6.
Exogenous expression of Rrp6 complements the synthetic lethality of rrp47∆ rex1∆ and rrp47∆ mpp6∆ mutants.
Yeast rrp47∆ rex1∆ and rrp47∆ mpp6∆ double mutants bearing plasmids with a URA3 marker and a wild-type copy of either the RRP47 (panels A-C) or MPP6 gene (panel D) were transformed with RRP6 constructs. Transformants were isolated on selective minimal medium and tested for growth in parallel on permissive minimal medium (left panel) and on medium containing 5 FOA (right panel). Plates were incubated at 30 °C for 3 days. The nature of the expression construct is indicated for each segment on the right.
Figure 7.
Growth assays of rrp47∆ rex1∆ and rrp47∆ mpp6∆ mutants.
Spot growth assays of rrp47∆ mpp6∆ (upper panel) and rrp47∆ rex1∆ (lower panel) double mutant isolates. The complementing construct is indicated on the left. 10-fold serial dilutions of standardised precultures were spotted on to selective solid medium and the plates were incubated at 30 °C. Plates were photographed after incubation for 3 days.
Figure 8.
Northern analyses of rrp47∆ rex1∆ and rrp47∆ mpp6∆ mutants.
Total cellular RNA was isolated from isogenic wild-type, rex1∆, rrp47∆ and mpp6∆ strains, and from rrp47∆ rex1∆ rrp47∆ or rrp47∆ mpp6∆ double mutants that are complemented by centromeric (cen) or 2 micron (2μ) plasmids expressing RRP47, MPP6 or RRP6 alleles. RRP6 constructs encoded either non-tagged or zz-tagged fusion proteins. RNA was resolved through 8% denaturing polyacrylamide gels and northern blot analyses performed, using probes complementary to the RNAs indicated on the right of each panel. (A-F) Analysis of rrp47∆ rex1∆ mutants. (G-N) Analysis of rrp47∆ mpp6∆ mutants. To compare the relative levels of both mature and 3’ extended forms of snoRNAs in the different strains in panels A-C, two images are shown from the same hybridisation. Dispersed bands labelled I-pA and II-pA represent snoRNAs that are polyadenylated after termination at sites I or II, respectively. Bands labelled 5S* and snR13* represent truncated RNAs.
Figure 9.
Rrp6 levels are not altered in mpp6∆ or rex1∆ mutants.
Western analyses were performed on extracts from strains that either carry a wild-type or a deletion allele of the MPP6 or REX1 gene and that express the zz-Rrp6 fusion protein. Blots were successively incubated with the PAP antibody and antibody specific to the Pgk1 protein. Expression levels of zz-Rrp6 in the mpp6∆ and rex1∆ mutants, relative to the level observed in the corresponding wild-type strain, are given at the bottom of the figure and are the average of three independent biological replicates.