3.3. Expression of the PP7 Coat Protein

Digest ~1 μg of the pMET-GFP-PP7-CP containing plasmid, transform into the verified haploid or homozygous diploid strains carrying the 24xPP7 cassette and NPC marker, and select for transformants on SC-Leu (see Notes 2 and 9).

Test multiple transformants for CP expression and the presence of fluorescently marked mRNPs using a fluorescence microscope under careful growth conditions in liquid culture using SC-Leu supplemented with 150 mg/liter of methionine (Met) and grown until early-log phase (O.D₆₀₀ of 0.1–0.3) at 25 °C. Single mRNP particles are often visible as is a general diffuse cell fluorescence from the free PP7-CP (Fig. la and Notes 10 and 11).

(a) Fluorescent images showing a cell in which PP7-CP has formed a bright aggregate. Raw and Laplacian filtered images are shown, each scaled to show the presence of the aggregate (asterisk) and single mRNP (arrow). Scaling to observe single mRNPs leads to difficulty in tracking single due to the presence of a bright foci. (b) Merged and registered images showing the bright PP7-CP aggregate in panel (a) persists in the same location through the imaging series, whereas single mRNPs are usually dynamic. Green channel = CP, red channel = nuclear pore complexes (NPCs)