Fig. 4

Yef3 is associated with mRNA in a ribosome-independent manner. Cells expressing TAP-tagged Yef3 were subjected to TAP purification and Yef3 associated mRNAs were analyzed. a Cells were lysed either in the absence (−) or presence (+) of 20 mM EDTA. (i) Protein samples from the input or the elution were subjected to western analysis with the indicated antibodies. The shorter bands detected in the Elution samples when αYef3 is used are consistent with cleavage of the TAP moiety by the TEV. Similarly, no signal is detected in the Elution when αTAP is used. (ii) Northern analyses with the indicated probes for RNA samples in the Elution samples from the minus EDTA (−) or plus EDTA (+) preparations. (iii) Quantitation of PMP1 isolated with Yef3-TAP by RT-qPCR. Samples were prepared either in the absence of EDTA (−EDTA) or its presence (+EDTA). To account for technical differences, an in vitro transcribed bacterial RNA (PHE, [51]) was spiked into each sample and its signals were used to normalize the PMP1 signals. (iv) RT-qPCR of PMA1 and PMP1, isolated in the presence of EDTA. b Yef3-TAP cells co-expressing either normal PMP1 (WT) or PMP1 with two stop codons immediately after the start codon (2× Stop) were subjected to TAP purification. RNA samples were subjected to RT-qPCR using primers for PMA1 and PMP1, and IP efficiency (Elution/Input) was determined. Values are normalized to the PMA1 signals