1. Targeted mRNA Degradation by Deadenylation-Independent Decapping
Gwenael Badis, Cosmin Saveanu, Micheline Fromont-Racine, Alain Jacquier 
Molecular Cell 
Volume 15, Issue 1, Pages 5-15 (July 2004)
DOI: /j.molcel

2. Figure 1 EDC3 Mediates RPS28B Autoregulation
(A) Transcriptome comparison of mRNAs from wild-type and edc3Δ strains performed using Affymetrix DNA microarrays (YG-S98). The arrow points to the RPS28B transcript signal.
(B) Transcriptome comparison of mRNA from wild-type and EDC3 overexpressing strains performed using Affymetrix DNA microarrays (YG-S98). The RPS28B and EDC3 transcript signals are indicated by arrows.
(C) Quantitation of the RPS28A and RPS28B transcripts in wild-type, edc3Δ, and EDC3 overexpressing strains. RPS28B and RPS28A mRNAs were analyzed by Northern blot using a radiolabeled oligonucleotide (GB096) that hybridizes with both RPS28B and RPS28A transcripts and were normalized with the ACT1 transcripts (see Experimental Procedures).
(D) Northern blot analysis of RPS28B transcript in wild-type and edc3Δ strains, transformed with no, centromeric (+cen RPS28B), or multicopy (+2 μ RPS28B) plasmids carrying RPS28B gene (plasmid pRS315/RPS28B + 3′ and pRS425/RPS28B + 3′, respectively, see Experimental Procedures). The RPS28B transcript levels normalized to ACT1 were determined using a Phosphorimager.
(E) Quantitation of the RPS28A and RPS28B transcripts in wild-type, rps28aΔ (LMA203), and rps28bΔ (LMA204) strains. RPS28A (left) and RPS28B (right) mRNAs were analyzed by Northern blot using a 5′ end-labeled oligonucleotide (GB096) that hybridizes with both RPS28B and RPS28A transcripts. The mRNAs were normalized relative to the snR35 transcripts hybridized with oligonucleotide MFR523 (see Experimental Procedures).
Molecular Cell  , 5-15DOI: ( /j.molcel )

3. Figure 2 EDC3 Influences the Stability of the RPS28B mRNA
The levels of the RPS28B mRNA were measured in a wild-type strain and in an edc3Δ strain by reverse transcription at different times after doxycycline-induced transcriptional repression. The plasmid pCM190/RPS28B + 3′UTR was introduced in strains deleted for the endogenous RPS28B gene in a wild-type or edc3Δ background (LMA204 and LMA225 strains). Doxycycline was added to the medium to turn off the expression of RPS28B. The resulting RPS28B transcripts and the endogenous RPS28A transcripts (used as a control) were analyzed by reverse transcription using oligonucleotide GB214 (see Experimental Procedures). These time courses were performed in triplicate and quantified with a Phosphorimager.
Molecular Cell  , 5-15DOI: ( /j.molcel )

4. Figure 3 The RPS28B mRNA 3′UTR Contains a cis Regulatory Element
(A) RPS28B 3′UTR is required to promote the EDC3-mediated regulation of RPS28B mRNA. The ratios of the RPS28B over RPS28A transcripts were measured by primer extension (plasmids pRS315/RPS28B + 3′UTR and pRS315/RPS28B − 3′UTR in LMA204 and LMA225 strains; see Experimental Procedures). These experiments were performed in triplicate and quantified with a Phosphorimager.
(B) The RPS28B 3′UTR can modulate the expression of a LacZ reporter gene. Plasmids pCM190/LacZ + 3′UTR and pCM190/LacZ − 3′UTR (as control) were transformed in strains deleted for the endogenous RPS28B gene (but with endogenous RPS28A gene) in a wild-type or edc3Δ background. β-galactosidase activity was measured by a colorimetric assay using ONPG as described (Miller, 1972).
(C) Alignment of the Saccharomyces cerevisiae RPS28B 3′UTR sequence with the corresponding sequences in Saccharomyces kluyveri, ZygoSaccharomyces rouxii (from Souciet et al., 2000), Saccharomyces bayanus, and Saccharomyces paradoxus (from Kellis et al., 2003). The alignments were performed using the program ClustalW (Thompson et al., 1994). Nucleotides conserved in all five sequences are schematized by black boxes; nucleotides conserved in three or four species are schematized in gray.
(D) Secondary structure of the conserved region of RPS28B 3′UTR. The conserved region of RPS28B (from 548 to 600 downstream of the ATG) corresponds to a putative secondary structure predicted using the Mfold program (Walter et al., 1994) represented in the 3′H scheme (left). Arrows represent changes in other yeast species. Five out of eleven loop or bulge positions carry a substitution. Among the 21 base pairs that define the structure, five show compensatory base changes and two show conservative changes. Only one base pair exhibits noncompensatory changes (shaded in gray). Nucleotide substitution found in Saccharomyces kluyveri are shown as underlined characters and those found in ZygoSaccharomyces rouxii are in italics. v3′H schematizes the variant version of the RPS28B regulatory hairpin used during the three-hybrid experiment: naturally occurring substitutions, shown in green, were introduced in order to allow expression from PolIII (see Results). mut1, mut2, and mut3 carry mutations (in red) within the terminal loop (mut1), the internal bulge (mut2), or the four apical base pair of the stem (mut3) of the RPS28B regulatory hairpin.
(E) The hairpin regulatory element 3′H is sufficient to promote RPS28B autoregulation. The ratios of RPS28B over RPS28A were measured in LMA204 and LMA225 strains. The level of different RPS28B transcripts (from pCM190/RPS28B + 3′UTR, pCM190/RPS28B − 3′UTR, and pCM190/RPS28B + 3′H) relative to the level of the RPS28A transcripts were analyzed as in Figure 3A.
(F) The hairpin regulatory element interacts with Rps28b. Yeast three-hybrid assays for binding of IRP (as control) (AD-IRP) and Rps28b (AD-RPS28B) to four RNA hybrids (in triplicates): IRE-MS2, v3′H-MS2 carrying the natural variant of the RPS28B regulatory hairpin, and mut1-MS2, mut2-MS2, or mut3-MS2 that carry the mutations described above. An interaction between the protein and the RNA determines activation of the LacZ reporter gene and thus a blue color for the colonies.
Molecular Cell  , 5-15DOI: ( /j.molcel )

5. Figure 4
The EDC3-Mediated Decay of RPS28B mRNA Requires the Presence of the cis Regulatory Hairpin
(A) Schematic organization of the RPS28B transcripts. The oligonucleotides used to target the RNase H cleavage or used as [32P]-labeled probes are shown by arrows.
(B) Analysis of poly(A) tail length of mutants of RPS28B 3′UTR mRNAs. The poly(A) tail average length of RPS28B with or without the conserved hairpin (+3′H or −3′UTR, see Experimental Procedures) was determined by RNase H treatment of mRNAs from wild-type (wt) or edc3Δ strains with GB096 primer and polyacrylamide Northern blotting using radiolabeled GB180 primer. Lanes marked dT correspond to wt samples treated with RNase H in presence of oligo dT18 to remove the poly(A) tail (as control). Phosphorimager profiles of the poly(A) tails from the mRNA expressed from constructs −3′UTR or +3′H, as indicated, in wt (EDC3, black line) or edc3Δ (gray lines) strains are shown in the right panels. The peak marked with an asterisk represents the profile of the mRNA after RNase H cleavage performed in the presence of oligo dT. The measures were quantified from the data shown in the left panel and normalized relative to the snRNA U4 probed with oligonucleotide MFR521 (data not shown).
Molecular Cell  , 5-15DOI: ( /j.molcel )

6. Figure 5
The RPS28B mRNA Regulatory Mechanism Is Independent of Deadenylation and Acts by Activation of Decapping
(A) Analysis of RPS28B mRNA poly(A) tail lengths after blocking transcription. The poly(A) tail average length of RPS28B transcript containing the hairpin (+3′H) was determined as described in Figure 4B. Total RNAs were extracted from cells taken at different time points after doxycycline-induced transcriptional repression. The plasmid pCM190/RPS28B + 3′H was introduced in EDC3 wild-type or edc3Δ cells with or without a galactose-inducible promoter upstream the endogenous CCR4 gene (wt, edc3Δ, GAL::CCR4, and GAL::CCR4/edc3Δ, corresponding to BMA64, LMA220, LMA328, and LMA329 strains, respectively; see Experimental Procedures). Strains GAL::CCR4 and GAL::CCR4/edc3Δ were grown in galactose medium and transferred to glucose medium for 20 hr. Doxycycline was added to the medium to turn off the expression of RPS28B.
(B) Phosphorimager profiles of the poly(A) tails from the mRNA expressed from constructs containing the 3′UTR hairpin (3′H) in different strains with or without EDC3 (wt, edc3Δ, GAL::CCR4, and GAL::CCR4/edc3Δ). The signals were quantified from the data shown in (A) and normalized relative to the snRNA U4 as in Figure 4B. mRNA half-lives were measured from quantifications on reverse transcription products as described in Figure 2.
(C) Poly(A) tails distribution of RPS28B transcripts when CCR4 is overexpressed. The poly(A) tail average length of RPS28B transcripts containing the hairpin (+3′H) was determined as described in Figure 4B. Total RNA was extracted from the GAL::CCR4 and GAL::CCR4/edc3Δ strains transformed by pCM190/RPS28B + 3′H and grown in galactose medium. The poly(A) tail average length of RPS28B transcript was determined as described Figure 4B, in CCR4 overexpressing conditions, with or without Edc3 (respectively CCR4 over in a wt or edc3Δ context). The corresponding Phosphorimager profiles are shown in the right panel.
(D) Poly(A) tails distribution of RPS28B transcripts when decapping is limiting. The plasmid pCM190/RPS28B + 3′H was introduced in a wild-type, edc3Δ, or dcp2Δ background (BMA64, LMA220, and LMA222). The poly(A) tail average length of RPS28B transcript was determined as described in Figure 4B. The corresponding Phosphorimager profiles are shown in the right panel.
Molecular Cell  , 5-15DOI: ( /j.molcel )

7. Figure 6
A Model for the Edc3-Mediated Autoregulation of the RPS28B mRNA
In the presence of excess Rps28, the protein binds to the conserved hairpin in the RPS28B mRNA 3′UTR and recruits the decapping machinery via an interaction with components of the decapping complex.
Molecular Cell  , 5-15DOI: ( /j.molcel )