Cytoplasmic Degradation of Splice-Defective Pre-mRNAs and Intermediates  Patricia J. Hilleren, Roy Parker  Molecular Cell  Volume 12, Issue 6, Pages 1453-1465 (December 2003) DOI: 10.1016/S1097-2765(03)00488-X

Figure 1 The GAL-UAS -WT, -C1, and -A257 Intron Reporter Genes Yield the Predictable Splice Products and 5′ to 3′ Decay Intermediates Transcription pulse-chase experiments (for growth conditions see the Experimental Procedures) of the wild-type strain (yRP1674) transformed with (A) GAL-mPGK1pG-WT (pRP1096), (B) GAL-mPGK1pG-C1, and (C) GAL-mPGK1pG-A257. In each panel, lane 1 represents molecular weight markers; lane 2 is the preinduced level of reporter RNA; lanes 3 and 4 are the 0′ time point (end of 5′ galactose induction) with (lane 3) or without (lane 4) oligo dT and RNaseH to remove the poly(A) tail; lanes 5–16 represent time points after glucose addition. A graphic on the right side of the panel shows the position of pre-mRNA, lariat intermediate, mRNA, and the products of 5′ to 3′ degradation. A cartoon at the top illustrates the relevant features of the reporter constructs. In brief, wild-type or mutant variants of the ACT1 intron are present in a mini version of PGK1pG (Decker and Parker, 1993) that carries a deletion of 1140 internal nucleotides (mPGK1pG). In this and subsequent cartoons, splice signals of the intron are designated in uppercase letters, and the specific mutations of each variant are indicated in lowercase. Exon1 is a fusion of PGK1pG and ACT1 exon 1 sequences that contains the 5′ UTR and start codon of mPGK1pG. Exon2 is derived fully from mPGK1pG. Expression of the GAL-UAS constructs is regulated by carbon source manipulations (see Experimental Procedures). Molecular Cell 2003 12, 1453-1465DOI: (10.1016/S1097-2765(03)00488-X)

Figure 2 The C1 and A257 Pre-mRNAs Degrade via the Cytoplasmic 5′ to 3′ mRNA Turnover Pathway Decay from steady-state experiments of the wild-type (yRP1674), dcp2Δ (yRP1358), xrn1Δ (yRP1764), rat1-1 (yRP1781), and upf1Δ (yRP1783) strains transformed with tetO7-mPGK1pG-C1 (pRP1104) or tetO7-mPGK1pG-A257 (pRP1106). Growth conditions are described in the Experimental Procedures. Lanes 1 and 2 correspond to RNA from the point at which doxycycline was added to the culture treated with (lane 2) or without (lane 3) oligo dT and RNaseH to remove the poly(A) tail. Lanes 3–8 show time points after doxycycline addition. Shown in the top half of each panel is the abundance of either C1 transcript (A) or A257 transcript (B) after hybridization with 32P-labeled oRP141. The half-life of the pre-mRNA is in parentheses. Pre-mRNA abundance was normalized to the level of 7S in each lane (bottom panel). On the right side of each panel is a graphic depicting the position of pre-mRNA. A cartoon at the top depicts the relevant features of the reporter constructs. Repression of transcription of the tetO7-regulated reporters is mediated by the addition of doxycycline (see Experimental Procedures). Molecular Cell 2003 12, 1453-1465DOI: (10.1016/S1097-2765(03)00488-X)

Figure 3 Analysis of the C303 Lariat Intermediate Reveals a Role for the Dbr1p in Initiating Degradation Transcription pulse-chase experiments of (A) wild-type (yRP1674), (B) rrp6Δ (yRP1762), (C) rrp44-1 (yRP1779), and (D) dbr1Δ (yRP1765) strains transformed with the GAL-mPGK1pG-C303 plasmid (pRP1098). In each panel, lane assignments are as in Figure 1. On the right side of each figure is a graphic depicting the position of pre-mRNA, lariat intermediate, mRNA, and/or the products of 5′ to 3′ degradation. The asterisk denotes a low level of spliced product that accumulates because of an alternate splice site choice (see Figure 1). In (D), the arrow indicates the position of a novel 3′-trimmed species that derives from the C303 lariat intermediate. The lower panels in each of the figures show an overexposure of a portion of the blot that highlights the fact that the 5′ to 3′ decay product has long poly(A) tails at early time points in the WT, rrp6Δ, and rrp44-1 strains that is absent in the drb1Δ strain. Additional signal seen at the top of (A) and (B) is due to hybridization of material stuck in the well. A cartoon at the top illustrates the relevant features of the GAL-UAS-regulated C303 reporter construct. Molecular Cell 2003 12, 1453-1465DOI: (10.1016/S1097-2765(03)00488-X)

Figure 4 The C303 Lariat Intermediate Degrades by Debranching Followed by Degradation by Xrn1p and/or Ski2p Decay from steady-state experiments of the (A) wild-type (yRP1674), (B) rrp6Δ (yRP1762), (C) rrp44-1ts (yRP1779), (D) xrn1Δ (yRP1764), (E) dbr1Δ (yRP1765), (F) dbr1Δ rrp6Δ (yRP1777), (G) dbr1Δ ski2Δ (yRP1778), and (H) rat1-1 (yRP1781) strains transformed with tetO7-mPGK1pG-C303 (pRP1105). In each panel, lane assignments are as described in Figure 2. Shown in the top half of each panel is the abundance of the C303 lariat intermediate after hybridization with 32P-labeled oRP141. Indicated in parentheses under each strain is the half-life of the pre-mRNA. Pre-mRNA abundance was normalized to the level of 7S RNA in each lane (bottom half of panel). On the right side of each panel is a graphic depicting the position of the lariat intermediate, the 3′-trimmed lariat intermediate (arrow), and the debranched intermediate that accumulates predominantly in the xrn1Δ strain. A cartoon at the top depicts the relevant features of the tetO7-regulated C303 reporter construct. Molecular Cell 2003 12, 1453-1465DOI: (10.1016/S1097-2765(03)00488-X)

Figure 5 In the Absence of Debranching, the Cytoplasmic Exosome Degrades the Lariat Intermediate (A) Transcription pulse-chase reactions of (A) dbr1Δ rrp6Δ (yRP1777) or (B) dbr1Δ ski2Δ (yRP1778) strains transformed with GAL-mPGK1pG-C303 (pRP1098). In each panel, lane assignments are as in Figure 1. Shown is the abundance of the C303 lariat intermediate after hybridization with 32P-labeled oRP141. On the right side of each panel is a graphic depicting the position of the lariat intermediate and in (A) the position of the 3′-trimmed species (arrow). A cartoon at the top of the panel illustrates the relevant features of the GAL-UAS-regulated C303 reporter construct. (B) Northern analysis of total RNA derived from strains transformed with tetO7-mPGK1pG-C303 (pRP1105). The top panel shows the abundance of the C303 lariat intermediate and its decay intermediates in the strains indicated using 32P-labeled oRP141. The bottom panel shows a reprobe of the membrane to detect the level of 7S RNA in each lane using 32P-labeled oRP100. Steady-state comparisons were made by normalizing the level of the C303 lariat intermediate and its decay products to the level of the endogenous 7S RNA levels and were subsequently expressed as a percentage of the level obtained in the wild-type strain. These RNAs were not oligo dT/RNaseH treated, and the doublet of lariat intermediate represents both poly(A)+ and poly(A)− species found at steady state. The arrow represents the discrete 3′-trimmed decay intermediate that is present in the absence of Rrp6p but absent when Ski2p is missing. A cartoon at the top depicts the relevant features of the tetO7-regulated C303 reporter construct. Molecular Cell 2003 12, 1453-1465DOI: (10.1016/S1097-2765(03)00488-X)

Figure 6 Dbr1p Functions in Quality Control during Pre-mRNA Splicing Decay from steady-state experiments of the wild-type intron (tetO7-mPGK1pG-WT) in the wild-type (yRP1674) and dbr1Δ (yRP1675) strains. In the dbr1Δ strain, the lariat intermediates represent ∼3% of total transcript at steady state (the 0′ time point), while these species are not detected in the isogenic wild-type strain (yRP1674). Relative to the short half-lives of the mRNA produced in both the wild-type and dbr1Δ strain (∼5′ each), these lariat intermediates are stable with a half-life of ∼25′. A cartoon at the top illustrates the relevant features of the tetO7 regulated wild-type intron reporter construct. Molecular Cell 2003 12, 1453-1465DOI: (10.1016/S1097-2765(03)00488-X)

Figure 7 A Model for Kinetic Competition among Processes that Impact the Fates of Yeast Pre-mRNA This cartoon depicts multiple processes during pre-mRNA maturation that may be in kinetic competition. What processes dominate will likely depend upon the idiosyncratic features either in cis or trans of any given pre-mRNA at each step. Molecular Cell 2003 12, 1453-1465DOI: (10.1016/S1097-2765(03)00488-X)