Proteomic characterization of messenger ribonucleoprotein complexes bound to nontranslated or translated poly(A) mRNAs in the rat cerebral cortex.
Receptor-triggered control of local postsynaptic protein synthesis plays a crucial role for enabling long lasting changes in synaptic functions, but signaling pathways that link receptor stimulation with translational control remain poorly known. Among the putative regulatory factors are mRNA-binding proteins (messenger ribonucleoprotein, mRNP), which control the fate of cytosolic localized mRNAs. Based on the assumption that a subset of mRNA is maintained in an inactive state, mRNP-mRNA complexes were separated into polysome-bound (translated) and polysome-free (nontranslated) fractions by sucrose density centrifugation. Poly(A) mRNA-mRNP complexes were purified from a postmitochondrial extract of rat cerebral cortex by oligo(dT)-cellulose affinity chromatography. The mRNA processing proteins were characterized, from solution, by a nanoflow reverse phase-high pressure liquid chromatography-mu-electrospray ionization mass spectrometry. The majority of detected mRNA-binding proteins was found in both fractions. However, a small number of proteins appeared to be fraction-specific. This subset of proteins is by far the most interesting because the proteins are potentially involved in controlling an activity-dependent onset of translation. They include transducer proteins, kinases, and anchor proteins. This study of the mRNP proteome is the first step in allowing future experimentation to characterize individual proteins responsible for mRNA processing and translation in dendrites.
Angenstein
Frank
F
Beckman Institute/Neuronal Pattern Analysis, University of Illinois, Urbana, Illinois 61801, USA. angenstein@ifn-magdeburg.de
Evans
Anne M
AM
Ling
Shuo-Chien
SC
Settlage
Robert E
RE
Ficarro
Scott
S
Carrero-Martinez
Franklin A
FA
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Greenough
William T
WT
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002540:Cerebral Cortex; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D046911:Macromolecular Substances; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D040901:Proteomics; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D012261:Ribonucleoproteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D046911:Macromolecular Substances; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein