Translational control is a crucial mechanism of gene regulation. Compared to transcriptional regulation, translational control of existing mRNAs allows for more rapid changes in cellular concentrations of the encoded proteins, due to relatively long, more than 2h, mRNA half-lives. In cells lacking active transcription such as oocytes and reticulocytes, translational control is often the only mechanism to regulate the synthesis of proteins. In highly polarized cells, such as neurons, oocytes or migrating cells, translational regulation is providing for local translation – when mRNA is expressed preferentially at specific parts of the cell, generating local pools of proteins.
Within this workshop, we will review the mechanisms of eukaryotic translation and translational control and focus on current methods that can be used to monitor and measure translation: RNA sequencing- and mass spectrometry-based (TRAP/Ribo-tag, ribosome profiling, proximity-specific ribosome profiling, PUNch-P, pulsed SILAC, BONCAT/QuaNCAT) and imaging-based (SUnSET/ribopuromycylation, puro-PLA/FUNCAT-PLA, TRICK, Nascent peptide imaging).

Content of the practical course
The practical part of the course will focus on the ribosome profiling protocol:

• Cell lysis
• Nuclease footprinting and ribosome recovery
• Isolation of footprinted fragments
• rRNA depletion
• Adaptor ligation
• Reverse transcription
• PCR amplification a
• Library purification and quality control with bioanalyzer
• Data analysis

Dr. Marina Chekulaeva: marina.chekulaeva@mdc-berlin.de Schließen