title: Analysis of plant ribosomes with asymmetric flow field-flow fractionation.
abstract: Ribosome profiling is a technique used to separate ribosomal subunits, 80S ribosomes (monosomes), and polyribosomes (polysomes) from other RNA-protein complexes. It is traditionally performed in sucrose gradients. In this study, we used asymmetric flow field-flow fractionation (AsFlFFF) to characterize ribosome profiles of Nicotiana benthamiana plants. With the optimized running conditions, we were able to separate free molecules from ribosomal subunits and intact ribosomes. We used various chemical and enzymatic treatments to validate the positions of subunits, monosomes, and polysomes in the AsFlFFF fractograms. We also characterized the protein and RNA content of AsFlFFF fractions by gel electrophoresis and western blotting. The reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ribosomes remained bound to messenger RNAs (mRNAs) during the analysis. Therefore, we conclude that AsFlFFF can be used for ribosome profiling to study the mRNAs that are being translated. It can also be used to study the protein composition of ribosomes that are active in translation at that particular moment.
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 27, 00014, Helsinki, Finland.
authors.forename: Leena
authors.initials: L
authors.lastname: Pitkänen
authors.forename: Päivi
authors.initials: P
authors.lastname: Tuomainen
authors.forename: Katri
authors.initials: K
authors.lastname: Eskelin
mesh_terms: D031521:Fractionation, Field Flow / Q000379:methods*; D010940:Plant Proteins / Q000302:isolation & purification; D018749:RNA, Plant / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000302:isolation & purification; D012270:Ribosomes / Q000737:chemistry*; D014026:Nicotiana / Q000737:chemistry* / Q000166:cytology; D014908:Triticum / Q000737:chemistry* / Q000166:cytology
chemical_list: D010940:Plant Proteins; D018749:RNA, Plant; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins