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What is rRNA?

Ribosomal RNA (rRNA) serves as the foundational framework of the ribosome, a vital organelle orchestrating protein synthesis. Unlike messenger RNA (mRNA), which carries genetic instructions for protein assembly, rRNA is a non-coding RNA that facilitates the actual process within the ribosome.

Derived from ribosomal DNA (rDNA), rRNA collaborates with ribosomal proteins to construct the ribosome’s two main components: the large subunit (LSU) and the small subunit (SSU). Remarkably, despite its non-coding nature, rRNA dominates the cellular RNA landscape, constituting approximately 80% of total RNA. This prevalence underscores its pivotal role in cellular function.

Comprising approximately 60% rRNA and 40% protein, ribosomes possess distinct sedimentation coefficients, denoting their size and composition. In prokaryotes, the subunits are denoted as 50S (large) and 30S (small), housing three classes of rRNA: 5S, 23S, and 16S. Conversely, eukaryotic ribosomes are designated as 60S (large) and 40S (small), hosting four classes of rRNA: 5S, 5.8S, 28S, and 18S. Notably, the 5.8S and 28S rRNAs collectively parallel the size and functionality of the prokaryotic 23S rRNA.

Table 1 The length of each rRNA

Type Size Large subunit (LSU) Small subunit (SSU)
Prokaryotic 70S 50S (5S: 120 nt, 23S: 2906 nt) 30S (16S: 1542 nt)
Eukaryotic 80S 60S (5S: 121 nt, 5.8S: 156 nt, 28S: 5070 nt) 40S (18S: 1869 nt)

The term “16S rRNA” denotes “16S ribosomal ribonucleic acid,” with “S” representing Svedberg units, a measure of sedimentation rate. This particular rRNA molecule serves as a critical constituent of the small subunit (SSU) found in prokaryotic ribosomes, as well as those in mitochondria and chloroplasts.

16S and 18S regions are commonly employed for microbial colony identification through amplicon sequencing, owing to their notable conservation and some variability. They serve as effective markers for discerning genera and species present within microbial populations, facilitating assessments of their relative abundance.

What Does rRNA Do?

Ribosomal RNA (rRNA) is instrumental in facilitating protein synthesis, a fundamental process crucial for cell function and survival. It accomplishes this through several key roles:

  • Catalyzing Protein Synthesis: rRNA acts as a catalyst for protein synthesis within the ribosome, ensuring the accurate assembly of amino acids into polypeptide chains.
  • Facilitating Ribosomal Functions: It plays a central role in ribosomal functions, including binding to messenger RNA (mRNA) to initiate translation and recruiting transfer RNA (tRNA) molecules carrying amino acids to the ribosome.
  • Structural Support: rRNA contributes significantly to the overall structure of the ribosome, accounting for approximately 60% of its weight. Its presence helps shape the ribosome and create specialized sites within it, such as the A (aminoacyl), P (peptidyl), and E (exit) sites.
  • Coordination of Protein Synthesis Steps: Within the ribosome, rRNA coordinates the sequential steps of protein synthesis. It guides the positioning of mRNA and tRNA molecules, facilitating the formation of peptide bonds between amino acids.
  • Binding to Ribosomal Proteins: rRNA contains binding sites for ribosomal proteins, aiding in the assembly and stability of the ribosome. These interactions contribute to the precise functioning of the ribosomal machinery.

Overall, rRNA plays a multifaceted role in the ribosome, ensuring the accurate and efficient synthesis of proteins essential for various cellular processes.

The Impact of rRNA on Sequencing

The quality of transcriptome sequencing is significantly influenced by several factors, notably RNA degradation during sample preparation, potential prokaryotic contamination, and interference from ribosomal RNA (rRNA). Typically, the RNAs of interest—such as mRNA, lncRNA, tRNA, and microRNA—comprise only a small fraction, typically 1% to 5%, of the total RNA pool. Some target RNAs may even represent an exceedingly minuscule proportion, as rare as one part in a million of the total RNA content, making their detection akin to searching for a needle in a haystack.

Compounding this challenge is the abundance of ribosomal RNA (rRNA), which constitutes a substantial portion, ranging from 80% to 98%, of total RNA. While rRNA serves as a vital component in protein synthesis within organisms, it proves extraneous and hinders the sensitivity of detecting low-abundance RNA in RNA sequencing (RNA-Seq), posing significant obstacles to studying target RNA.

Consequently, a primary strategy in RNA research involves enriching sequencing data for target RNAs by selectively removing the abundant, nonessential rRNAs. Despite its pivotal role in protein synthesis, rRNA takes a backseat to messenger ribonucleic acid (mRNA) in genetic regulatory studies and RNA-Seq technologies. In the realm of microbial single-cell transcriptome sequencing, the absence of PolyA tails in prokaryotic mRNAs precludes the adoption of eukaryotic mRNA capture methods, necessitating rRNA depletion as a preferred approach.

Mitigating rRNA Interference in Transcriptome Sequencing: Strategies and Implications

The quality of transcriptome sequencing is influenced by several key factors, including the extent of RNA degradation during sample preparation, potential prokaryotic contamination, and interference from ribosomal RNA (rRNA). Consequently, a major strategy in RNA research involves selectively removing high levels of unwanted rRNAs to significantly enhance the proportion of sequencing data dedicated to target RNAs. It includes PolyA Enrichment and RNase H Method.

Table 2 Methods for rRNA removal

Classification of Methods mRNA Enrichment rRNA Removal
Method Name PolyA Enrichment Method RNase H Method
Principle RNA with Poly(A) tail is enriched by hybridization with oligo(dT) molecules on the surface of beads after denaturation to remove the hierarchical structure DNA probe binds to target RNA, and RNase H digests the RNA strand on the RNA-DNA probe complex; unhybridized RNA sample remains undigested
Applicable Samples RNA samples with Poly(A) tails RNA of known sequence
Advantages – Commonly used, simple, and cost-effective method for Poly(A)-tailed RNAs
– Shown to be effective for various species
– Relatively inexpensive and simple to perform
Disadvantages – Not applicable to prokaryotes
– Requires high RNA integrity; mRNA with broken Poly(A) tail cannot be enriched
– Cannot enrich non-Poly(A) tailed RNAs such as lncRNA, circRNA, etc.
– Different species require different probe designs
– Risk of RNA degradation by RNase H

Enriching target RNAs through mRNA enrichment or removal of irrelevant rRNAs presents a strategic approach that not only lowers sequencing costs but also enhances the success rate and efficiency of RNA research. These methods are versatile across various species and sample types, each offering distinct advantages and disadvantages. Furthermore, they have led to the development of various commercialized products tailored to different research needs.

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