Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

Abstract

AbstractmRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed "anota" algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

nanoCAGE reveals 5′ UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs

“…In polysome-profiling, translation efficiency is directly determined by separating efficiently (commonly defined as mRNAs associated with more than three ribosomes) and not efficiently translated mRNAs (defined as mRNAs associated with three or less ribosomes) (Gandin et al 2014). In contrast, with ribosomeprofiling translational efficiency is inferred based on the number of RNA sequencing reads corresponding to ribosome-protected fragments (Gandin et al 2014;Ingolia 2014;King and Gerber 2014). TOP mRNAs are highly expressed (Fig.…”

“…Polysomeprofiling was carried out as described (Gandin et al 2014). RTqPCR was performed as previously described (Miloslavski et al 2014).…”

“…RTqPCR was performed as previously described (Miloslavski et al 2014). Fractions were collected as described in Gandin et al (2014), and RNA was extracted using TRIzol according to the manufacturer's instructions. RT and qPCR were performed by SuperScript III Reverse Transcriptase and Fast SYBR Green Mastermix (Invitrogen), respectively.…”

IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

“…Cells were incubated with cycloheximide (Santa Cruz Biotechnology) (100 μg/mL) in phase 2 media for 5 min and washed twice in cold PBS supplemented with cycloheximide (100 μg/mL) before lysis using 20 mM Tris·HCl (pH 8.5), 1.5 mM MgCl 2 , 140 mM KCl, 0.5 mM DTT, 0.5% Nonidet P-40, 200 units/mL ribonuclease inhibitor, and 0.1 mM cycloheximide as previously described (16). Sucrose gradient sedimentation analysis was performed by standard methods (58). Samples were loaded onto 10-50% gradients in 20 mM Tris·HCl (pH 8.0), 140 mM KCl, 5 mM MgCl 2 , 0.5 mM DTT, and 1 mg/mL cycloheximide buffer, and spun in a Beckman Coulter SW 41 Ti rotor at 40 krpm for 3 h at 4°C.…”

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of <em>Drosophila</em> Embryos

“…Among newly developed approaches polysome profiling by fractionation was described as a way to capture actively-translating RNA 11 . This method based on sucrose gradient centrifugation allows the selection of polysome fraction and determination of mRNA translated genome-wide when analyzed with microarrays or deep sequencing.…”