RNA-Seq has been used consistently for years as a way to determine gene expression by correlating mRNA levels to protein levels. However, the actual translation process in vivo cannot be completely captured by this method. This is because each mRNA molecule isn’t necessarily translated into protein by ribosomes. Ribosome Profiling was developed to help complete this picture.

In this blog, we’ll go over what Ribosome Profiling is, some real-world applications, a typical workflow, and how Genohub can help you with your Ribo-Seq project.

What is Ribosome Profiling?

In order to synthesize proteins, cells transcribe mRNA from DNA and then translate proteins from mRNA. Many researchers who want to study this gene expression process have used RNA-Seq, which provides data on the relative levels of mRNA within a cell. While the levels of specific mRNA often do correlate with the levels of particular proteins, standard RNA-Seq cannot provide actual data regarding gene regulation at the translational level. This is where Ribosome Profiling (Ribo-Seq) comes in.

Ribo-Seq is a sequencing method that uses specific ribosome-protected mRNA fragments (RPFs) to determine the mRNAs that are actively being translated in vivo. This snapshot can then be compared to parallel RNA-Seq done for the transcriptome to reveal the positions and amounts of ribosomes on any specific mRNA.

What are the applications of Ribo-Seq?

Ribo-Seq can help identify alternative mRNA translation start sites, confirm annotated open reading frames (ORFs) and upstream ORFs that may be involved in translation regulation, the distribution of ribosomes on an mRNA and the rate at which ribosomes decode codons. As Ribo-Seq can provide data about gene expression, protein synthesis and protein abundance, it can be useful in almost every type of research, including research on cancer, autoimmune disease, heart disease, neurological disorders, and psychiatric disorders.

The following are examples where Ribo-Seq was used in different types of research.

• Scheckel et al. used Ribo-Seq in combination with another technique to discover that aberrant translation within the glia only may be enough to cause severe neurological symptoms and may be a primary driver of prion disease.

• In this paper, the authors summarize multiple studies where Ribo-Seq was used to identify novel genes within plants that could be useful to increase yield through biotic and abiotic stress tolerance if manipulated.

• In this article, Ribo-Seq was used to reveal translated sequences within long noncoding RNAs and to identify other micropeptides within two herpesviruses, human cytomegalovirus and Kaposi’s sarcoma-associated herpesvirus. Understanding viral gene regulation and other aspects of the proteome are important for understanding their life cycle and identifying epitopes they may present for immune surveillance.

What is the typical Ribo-Seq workflow?

The typical Ribo-Seq workflow begins with collecting and preparing the lysate. First, the cells or tissue samples are harvested and flash-frozen to halt translation. Then, the samples are resuspended in a lysis buffer that includes a salt to stabilize the ribosomes, detergent to puncture the cell membrane, a deoxyribonuclease to degrade genomic DNA, a translation-inhibiting drug to halt the ribosome, and a reducing agent to stop oxidative compounds from interfering with RNA. After lysis, ribonucleases are added to digest the RNA that is not protected inside of the ribosomes. These fragments are called RNA protected fragments (RPFs). Then size selection is performed to identify the ~28 nucleotide RPFs on a gel, and RNA extraction is extracted. Any contaminating rRNA is removed, the RPFs are reverse-transcribed to cDNA, amplified by PCR and then made into libraries that are sequenced.

The data analysis done will ultimately depend on the researcher’s personal aim, but in general, ribosome profiling mapping would include data QC, demultiplexing and then removal of adapter sequences and any remaining rRNA contaminants. The samples would then be aligned to an annotated genome/transcriptome and then counts of the number of reads aligned to each gene would be obtained. These mapped RPFs can then be visualized and compared with what other researchers have done. More specific analysis can include uORF detection, differential gene expression, global translation rates, ribosome stalling, and codon decoding rates.

Where can I get help with my Ribo-Seq project?

As of now the Illumina kit for Ribo-Seq, TruSeq Ribo Profile or ART-Seq, has been discontinued. There is a commercially available all-inclusive library preparation kit, called LACESeq by IMMAGINA Biotechnology. However, Ribo-Seq sample and library preparation is so complex and sample-specific that many labs have their own protocols optimized for their specific samples and then use their favorite commercial small RNA-Seq kit for the last part of library prep. For labs that don’t focus on this type of work, optimizing such a protocol can be very time-intensive and expensive.

Genohub’s Ribo-Seq partners are experts in every step of the Ribo-Seq process, from lysis to custom data analysis, including preparing and running RNA-Seq libraries in parallel, allowing for the measurement of translation efficiency. Our in-network partners also have experience in isolating ribosome-bound mRNA from many different types of samples, including bacteria and eukaryotic cells, and animal and plant tissue. Their proprietary optimized Ribo-Seq protocols means they routinely produce high-quality libraries efficiently and effectively. All you would have to do is provide your frozen cell or tissue samples and let us do the rest.We will be with you every step of the way, from extraction to data analysis! Get started today by letting us know about your Ribo-Seq project here: https://genohub.com/ngs/ .