A comparison of Polysome Profiling, RNC-Seq and Ribo-Seq (Part Two)

2. RNC-Seq

Translating mRNA full-length sequencing (RNC-SEQ) shows a unique advantage that effectively addresses this issue. The cell lysate was loaded on a 30% sucrose pad and all the ribosomal-related translated mRNA and free mRNA and other cellular components were separated by ultracentrifugation. Ultracentrifugation can deposit RNC. RNC-mRNA can be recovered from the granulated RNC, effectively avoiding the interference of high concentration of sucrose, which is beneficial to downstream research. Next-generation RNC-mRNA sequencing technology reveals the full-length information of translated mRNAs, including the abundance and type of mRNAs. By optimizing the centrifugation and sucrose buffer, the recovery of RNC can reach 90%. Under the condition of appropriate buffer, RNC still maintains translation activity. The technical difficulty of RNC-SEQ lies in the separation of complete RNC. The fragility of RNC leads to the dissociation of ribosomes and the breakage / degradation of mRNA, which leads to the biased analysis of RNC-mRNAs.

3. Ribo-Seq

Ribosome map (Ribo-seq), first published by Science in 2009 in Ingolia et al, which studied translation from another perspective. Treating cell lysates with low concentrations of ribonuclease (RNase) degrades mRNA, except for ribosome-protected RNA fragments. Next-generation sequencing (NGS) was used to analyze 22–35 nt mRNA fragments (ie, ribosome footprints: RFPs), which correspond to ribosome-protected fragments (RPFS) to reveal the location and density of ribosomes. Based on positional information, the distribution and density of ribosomes on each transcript, information such as the start codon position (including non-ATG start), codon usage bias, upstream ORFs (uORFs), and translation pause landscape can be inferred. These aspects cannot be studied by other translation methods. In 2016, an optimized Ribo-seq method-super-resolution ribosome profiling was developed, which was able to observe a strong global 3nt periodicity in a single transcript. In addition, this method improves Ribo-seq’s ability to reveal small ORFs (sORFs) and unannotated new coding regions, possibly encoding proteins from annotated non-coding RNA and pseudogenes. The ribonuclease used in the ribosome map was also taken into account. It was found that ribonuclease T1 is the enzyme that can best maintain the integrity of the ribosome, and also can convert the multimer into a single nucleosome. A variant of Ribo-seq is translation complex profile sequencing (TCP-seq). Fast-cooled yeast cells were cross-linked with formaldehyde to cause their translation complexes with mRNA to stagnate in their natural positions, and then subjected to RNase digestion. Then sucrose gradient ultracentrifugation was used to separate whole ribosomes and small subunits (SSU), and RNA fragments of up to 250 nt in these fragments were sequenced. Natural distribution maps were obtained at the beginning, extension and termination stages of translation. This method can observe the SSU footprint on the 5 ‘untranslated region (UTR) of mRNA and capture the position of any type of ribosome-mRNA complex at various stages of translation.

However, Ribo-seq experiments are complicated and expensive. Compared to RNC-SEQ, it requires a large number of cells as a starting material. Since Ribo-seq of all species requires depletion of hybrid-based rRNA, Ribo-seq is usually limited to several model organisms. For many other species, especially the wide variety of bacteria, Ribo-seq is difficult to perform due to the lack of rRNA probes, and expensive customization of rRNA probes seems to be the only option. Many other factors affect the number of RFPs, such as pseudo RPF. Since Ribo-seq mainly analyzes coding sequences (CDS), where ribosomes bind to mRNA. Untranslated regions (UTRs) highly related to translation regulation cannot be effectively analyzed. In addition, Ribo-seq often generates many “RFPs” that are aligned to non-coding RNA, indicating a significant false positive rate.

Another disadvantage is the short RFP length (24–26 nt for prokaryotes and 28–30 nt for eukaryotes), which is limited by the size of the ribosome and cannot be extended further. In order to obtain sufficient coverage of moderately abundant mRNAs, the amount of sequencing needs to be expanded (usually more than 100 million reads per sample), which means that sequencing and computational costs are high. Nevertheless, many translation events, especially the splice junctions of splice variants and circular RNAs, remain difficult to cover. The stitching alignment algorithm performs poorly at detecting connection points in these short reads. In contrast, the full-length RNC-seq sequence is the sequence of the entire mRNA; therefore, longer read lengths are suitable. Longer reads result in almost complete coverage of most translated mRNAs, including low-abundance mRNAs. This allows efficient detection and quantification of ligation, for example, translation of various splice variants of BDP1 and BRF1 and translation of circular RNA CircLINC-PINT, which are almost impractical using Ribo-seq.

It is worth emphasizing that the density of RFP does not represent translation activity. The RFP density is directly proportional to the translation initiation rate and inversely proportional to the elongation. If translation is completely stalled on a certain mRNA, RFP will be highly enriched in that mRNA, but translation activity will be zero.

4. TRAP-Seq

Inada et al. reported ribosome affinity purification (RAP) or translating RAP (translating RAP: TRAP). The large ribosomal subunit protein Rp25p produced under the control of a tissue-specific promoter and a fusion affinity tag (such as polyhistidine, green fluorescent protein (GFP), etc.) is used at the C-terminus. These ribosomes are then affinity purified (beads or columns) and isolated from ribosomes of other cell types. TRAP-SEQ specifically enriches RNC-mRNA in difficult-to-separate samples isolated ribosomes cannot be contaminated with non-ribosomal mRNPs co-precipitated with ribosomes because TRAP-SEQ does not use ultracentrifugation. TRAP-SEQ has its unique advantages in isolating translated mRNA from specific cell types in complex tissues. However TRAP-seq requires a stably transfected cell line to produce labeled ribosomal proteins. When applied to plants and animals, it is inevitable to build stable transgenic organisms. This is time-consuming and expensive, and it is not suitable for those that have not yet been established Species that stabilize transformation methods. In addition, overproduction of labeled ribosomal proteins has the potential to alter the structure and properties of these ribosomes. As a result, the system is no longer under physiological conditions; careful evaluation should be performed before all conclusions are applied to general scenarios.