Figure 3.

Translation Repression of the MAA7 Gene Is Greatly Diminished in a C. reinhardtii Mutant Deleted for the EXP5 Ortholog (Encoded by Cre10.g420400) and Does Not Involve Transcript Deadenylation.

(A) Growth and survival of the indicated strains on TAP medium without (left) or with (right) 7 μM 5-FI. Maa7-IR44s(exp5), Maa7-IR44s strain containing a deletion of the Cre10.g420400 gene.

(B) Immunoblot analysis of TSβ abundance. Coomassie blue staining of an equivalent gel is shown as a control for similar loading of the lanes (bottom panel).

(C) RNA gel blot analysis of agarose gel–separated total RNA samples sequentially hybridized with ³²P-labeled PCR products corresponding to the coding sequence of MAA7 (top panel), to evaluate the degree of mRNA reduction, or the coding sequence of ACT1 (bottom panel), to assess the amount of sample loaded per lane.

(D) Detection of siRNAs in the Maa7-IR transgenic strains. Total cell RNA was separated in a 15% denaturing polyacrylamide gel, electroblotted onto a nylon membrane, and hybridized with the MAA7 3′ UTR sequence (top panel). The same filter was reprobed with the U6 small nuclear RNA sequence (bottom panel) as a control for equivalent loading of the lanes. nt, nucleotides.

(E) Analysis of polyadenylated tail lengths of the MAA7 and ACT1 transcripts in the indicated strains. Poly(A) tail lengths were examined using a G/I tailing protocol and RT-PCR assays (see Supplemental Figure 4 online). Reactions were performed as described in the Methods in the presence (+RT) or absence (−RT) of reverse transcriptase.