From Cells to Data - The Essential Steps in Ribosome Footprinting for Translation Research

Ribosome footprinting technology, a powerful method for studying protein synthesis and translation regulation, enables researchers to map the interaction between ribosomes and mRNA in living cells. This technique provides insight into the dynamics of translation by identifying the regions of mRNA that are protected by ribosomes during translation. The process involves several crucial steps, including cell processing, RNA extraction and purification, denaturation and electrophoresis, cDNA synthesis and sequencing library construction, high-throughput sequencing, and data analysis. Each of these steps contributes to generating high-resolution ribosome profiling data that can elucidate the translation mechanisms within cells. This article outlines the essential procedures involved in ribosome footprinting.

Cell Processing and Lysis: Freezing Translation Initiation

The first step in ribosome footprinting is the processing and lysis of cells. To ensure the capture of ribosome-associated mRNA segments, translation initiation must be halted immediately, typically by using translation inhibitors or freezing the cells.

• Translation Inhibition with Antibiotics or Freezing

Translation inhibitors such as streptomycin or cycloheximide are commonly used to halt translation. Streptomycin blocks ribosome translocation, preventing the elongation of the peptide chain, while cycloheximide inhibits the elongation process by preventing the translocation step. Alternatively, cell freezing in liquid nitrogen can rapidly arrest translation, preserving the interaction between ribosomes and mRNA at the time of freezing.

• Cell Lysis and Protection from RNA Degradation

After treatment, cells are lysed in a buffer containing low concentrations of RNAse inhibitors to prevent degradation. The use of a lysis buffer with a combination of salts and detergents ensures the release of cytoplasmic contents while maintaining the integrity of the ribosome-mRNA complex. To prevent RNA degradation during the lysis process, RNAse I or similar nucleases can be used at controlled concentrations to selectively degrade exposed RNA segments that are not protected by ribosomes. This ensures that only ribosome-protected fragments (RPFs) remain intact for downstream analysis.

Ribosomal RNA (rRNA) Removal: Isolating Ribosome-Protected Fragments

Ribosome footprinting focuses on the regions of mRNA protected by ribosomes. To achieve this, it is necessary to remove the abundant ribosomal RNA (rRNA) that constitutes a significant portion of the total RNA.

• Techniques for rRNA Depletion

Several methods can be employed to remove rRNA, including ultracentrifugation and gel electrophoresis. Ultracentrifugation separates ribosomes from other RNA species based on size, enabling the isolation of ribosome-bound mRNA fragments. Alternatively, rRNA can be removed by gel electrophoresis, where RNA samples are fractionated based on size, with rRNA being separated from the smaller mRNA fragments.

These techniques leave behind the RPFs, which are the mRNA segments protected by ribosomes. These fragments are the key to understanding translation dynamics, as they represent the regions of mRNA actively engaged in protein synthesis.

RNA Extraction and Purification: Ensuring High-Quality Samples

Once the rRNA has been removed, the next step is RNA extraction and purification. This step is crucial for ensuring the integrity and quality of the mRNA samples used in subsequent analysis.

• RNA Extraction

To extract the RNA, Trizol reagent or commercial RNA extraction kits are commonly used. Trizol reagent facilitates the separation of RNA from proteins and lipids in a single-step process. After the addition of chloroform and centrifugation, the RNA can be isolated in the aqueous phase. The integrity of the RNA is verified by running an aliquot on an agarose gel, ensuring that the samples do not exhibit degradation and are of high quality for subsequent analysis.

• RNA Purification

After extraction, RNA samples must be purified to remove contaminants such as proteins, lipids, and other impurities. This is typically done through column-based purification methods or by using ethanol precipitation. The final purified RNA will be free from these contaminants and ready for further analysis.

Denaturation and Electrophoresis: Fragment Separation and RNA Integrity

Denaturation and electrophoresis are key steps in separating the RNA fragments and ensuring that they are in the correct conformation for subsequent analysis.

• RNA Denaturation

To ensure uniform RNA separation, the RNA samples are denatured by heating them at temperatures between 60-80°C. This process unfolds the RNA secondary structures, ensuring that the molecules are in a linear form before electrophoresis. After denaturation, the samples are rapidly cooled on ice to prevent the formation of secondary structures that could interfere with subsequent analyses.

• Electrophoresis on Polyacrylamide Gel

The denatured RNA samples are then subjected to polyacrylamide gel electrophoresis (PAGE). This technique separates RNA fragments based on their size, with smaller fragments migrating faster through the gel matrix. SDS-PAGE or urea-PAGE may be used, depending on the nature of the RNA samples. The goal of this step is to resolve the different RNA fragments, including the RPFs, which will later be isolated for sequencing.

Fig 1 Assessment of different RNA conformations by native PAGE.¹

Staining and Cutting: Isolating Ribosome-Associated Fragments

Following electrophoresis, the RNA fragments are visualized using a staining technique. This step is crucial for identifying and isolating the ribosome-protected RPFs.

• Gel Staining

SYBR Green or other RNA-specific dyes are commonly used to stain the RNA fragments. The gel is stained, and the bands corresponding to the RPFs are visualized under a UV transilluminator. The RPFs typically appear as distinct bands, which represent the mRNA regions covered by ribosomes.

• Cutting the Targeted Fragments

Once the target fragments have been identified, the appropriate regions of the gel are excised. These fragments, which contain the ribosome-protected mRNA segments, are then extracted from the gel and prepared for further processing, such as cDNA synthesis.

cDNA Synthesis and Sequencing Library Construction: Preparing for High-Throughput Sequencing

The isolated RNA fragments are then reverse-transcribed into cDNA (complementary DNA) to prepare them for high-throughput sequencing.

• Reverse Transcription and cDNA Amplification

The RNA fragments are reverse-transcribed using reverse transcriptase and random primers. A key enzyme in this process is T4 RNA ligase, which can ligate the RNA fragments to adapters or primers necessary for sequencing. After the cDNA has been synthesized, the resulting cDNA fragments are amplified using PCR (polymerase chain reaction), which increases the quantity of the cDNA and prepares it for sequencing.

• Sequencing Library Construction

Once amplified, the cDNA is prepared into a sequencing library. This involves ligating sequencing adapters to the ends of the cDNA fragments, allowing them to be read by sequencing platforms such as Illumina. The final library is ready for high-throughput sequencing.

High-Throughput Sequencing and Data Analysis: Mapping Ribosome Footprints

With the sequencing library prepared, high-throughput sequencing technologies are used to obtain the sequence data of the ribosome-protected RNA fragments.

• Sequencing on Illumina Platforms

Illumina sequencing platforms are widely used for ribosome footprinting due to their high throughput and accuracy. The sequencing process generates millions of short reads, which correspond to the ribosome-protected mRNA fragments.

• Data Alignment and Ribosome Profiling

The sequencing reads are then aligned to a reference genome or transcriptome using specialized bioinformatics tools. This alignment allows researchers to pinpoint the exact locations of ribosome footprints on the mRNA. Further analysis can reveal important information such as translation elongation rates, start codon positions, and translation pausing regions, providing insight into the dynamics of translation regulation.

Fig 2 The flowchart of computational pipeline for analyzing ribosome footprint data.²

Validation and Follow-Up Experiments: Confirming Findings

After obtaining the ribosome profiling data, it is essential to validate the findings and potentially explore the functional implications of specific genes or translation regulation mechanisms.

• Validation Using RT-qPCR

To confirm the results obtained from ribosome footprinting, RT-qPCR can be used to validate the expression levels of key genes. This technique allows for the quantification of specific mRNA sequences and can verify the results of ribosome profiling.

• Follow-Up with Functional Studies

Additionally, researchers may use other experimental approaches, such as gene editing, to manipulate specific genes or sites identified in the ribosome profiling data. These follow-up experiments can help elucidate the functional role of specific translation events or regulatory mechanisms in cellular processes.

Conclusion

Ribosome footprinting technology provides a detailed and high-resolution view of translation regulation, from translation initiation to termination. By following the outlined steps—from cell processing and RNA extraction to sequencing and data analysis—researchers can uncover critical insights into protein synthesis mechanisms. The ability to study translation in vivo offers opportunities for a deeper understanding of gene regulation, with implications for cancer research, developmental biology, and beyond.

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References

1. Edelmann, Franziska Theresia, Annika Niedner, and Dierk Niessing. "Production of pure and functional RNA for in vitro reconstitution experiments." Methods 65.3 (2014): 333-341. Distributed under the Open Access license CC BY 3.0, without modification.
2. Zhong, Yi, et al. "RiboDiff: detecting changes of mRNA translation efficiency from ribosome footprints." Bioinformatics 33.1 (2017): 139-141. Distributed under the Open Access license CC BY 4.0, without modification.