Figure 1.
Schematic illustration of the cytoplasmic mRNA decay complex.
Decaysome internal associations and associations to the mRNA, which are used in our model, are depicted with broken lines. The process in which each factor participates is as follows (see parenthesized numbers): (0) – translation, (1) – deadenylation, (2) – decapping, (3a) –5′ to 3′ degradation, (3b) –3′ to 5′ degradation, (4) coordinator.
Table 1.
Summary of in vivo observations regarding Xrn1p and Dcp2p import dependencies.
Figure 2.
mRNA Decay and Nuclear Import Process.
(A) The mRNA Decay and Nuclear Import Process is in-zoomed into three sub-processes; Recruitments and Deadenylation, Decapping and Degradation and Decaysome Import. (B) The corresponding, automatically-generated textual Object-Process Language (OPL) paragraph that reflects textually what the diagram represents graphically.
Table 2.
In-silico DFs import results for permutations on DFs interactions, tested for Xrn1D208A mutation.
Table 3.
In-silico DFs import results and processes activation when deleting one factor/domain at a time.
Figure 3.
Experimental validation of our model-based hypothesis shows that some mRNA decay factors are associated with both full length mRNA and its degradation intermediate.
(A) Cells expressing RPL30pG (“Construct B” in [18]), whose 3′ non-coding region contains (G)18 tract, and carrying the indicated TAP-tag in place of the natural genes were used. The indicated TAP-tagged proteins were affinity purified using two affinity steps as described previously [19], under conditions that minimized RNA degradation in vitro. The RNA was extracted and subjected to Northern analysis [24]. The probe contained the (C)18 and flanking regions; it was hybridized under stringent conditions (75°C) and, except for one non-specific band (marked by asterisk), was highly specific [18]. Accumulation of the degradation fragment (denoted “Frag.”) was due to the (G)18 tract that blocked the 5′ to 3′ exonuclease activity of Xrn1p [20, 21]. The Fragment therefore spans from the (G)18 tract till the 3′ end of the RNA (88 b including the G tract). Input was obtained by subjecting total RNA (before IP) to Northern analysis; IP are the affinity purified samples. Percent of “Fragment” from FL was determined by measuring radioactivity using the PhosphImager technology. All lanes were taken from the same gel. (B) Cells expressing MFA2pG, whose 3′ non-coding region contains (G)18 tract [20, 21], and carrying RPB4-TAP in place of RPB4, or carrying untagged RPB4 (“No TAP”) were used. TAP purification followed by Northern analysis was done as in A. The affinity purification of Rpb4-TAP was performed three times (twice for examining RPL30pG [A] and once for examining MFA2pG [B]), and those of Pan2 and Lsm1 repeated twice. Whereas the % of “Fragment” from full length mRNA was different in different experiments (it is highly sensitive to the strain background and to the nature of the mRNA, and somewhat also to the proliferation conditions), in all cases, the proportion of Fragment in the immunoprecipitated material was similar to that in the input material, as shown in this figure. FL – full length. Frag. – “Fragment”. ND = not determined.
Figure 4.
Experimental validation of model’s predictions shows that import of some decay factors is independent of Xrn1p exonuclease activity.
XRN1 (WT) PAB1-GFP xpo1-1, mex67-5 cells, or xpo1-1, mex67-5, Δxrn1 cells expressing xrn1D208A-GFP and RFP fusion of the indicated DFs were proliferated at 24°C and then shifted to 37°C for 1 h; images were taken as previously described [10]. (A) Representative images of WT cells expressing the indicated proteins after 1h incubation at 37°C. Pab1-GFP, whose export is dependent on Xpo1p and Mex67p, serves as a nuclear marker, as described in [10]. Arrows point at examples of nuclei carrying both fluorescent proteins. All factors were cytoplasmic at 24°C ([10] and not shown) (B) Percentage of cells with nuclear localization of the indicated DF was determined, as described previously [10]. Mean values ± SD are shown. P-values of any pairwise difference that was <0.05 is indicated.