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Small RNA Modifications

The Challenges and Solutions of Studying Modified Small RNAs

 

 

Small RNA Modifications: Integral to Functions and Diseases

Molecular Mechanisms of Small RNA Modifications

The Challenges and Solutions of Studying Modified Small RNAs

 

Profiling small RNA modifications is a key step in studying the increasingly important epitranscriptomics of the small RNA classes. However, not until recently, the methodologies and techniques were lacking or very limited in capabilities. With the new innovative array based technologies, the high throughput, sensitive, accurate, and modification stoichiometry capable small RNA profiling is now available to biological and clinical research labs to advance the new study of small RNA epitranscriptomics and clinical applications.

 

The challenges of profiling small RNA modifications

Although sequencing has been used for small RNA profiling, the influence of RNA modifications on the sequencing quantification has largely been ignored. Various RNA modifications, such as m1A, m3C and m1G, do interfere with the reverse transcription reaction during sequencing library construction, thereby making accurate quantification of small RNAs and especially their modifications impossible. For example, the presence of m1A in the TΨC loop blocks the proceeding of reverse transcriptase during cDNA synthesis. Most of the small RNA sequencing data were obtained using the library construction methods above. Consequently, the data could be misleading for modified small RNAs.

Also, small RNA profiling by small RNA-seq requires multiple PCR amplification steps, which incurs significant quantification bias/inaccuracies and therefore necessitates the use of independent, orthogonal methodologies.

In practice, most sequencing based method for modification studies requires large amount of input materials (> 100 ug total RNA), precluding many studies with only limited sample amounts.

Furthermore, small RNA-seq commonly uses Reads Per Million RNA reads (RPM) for normalization and to represent the relative RNA abundances in the sample. However, RPM depends on the composition of the small RNA population in a sample. A change in one small RNA’s RPM will adjust all the other small RNAs’ values even their actual absolute expression levels are not changed.

In order to identify and quantify the full spectrum of modified-small RNAs with high sensitivity and accurate stoichiometry, there is a need for overcoming the limitations of the sequencing-based approaches and developing non-sequencing-based methods.

 

Techniques to quantify small RNA post-transcriptional modifications

Arraystar small RNA modification profiling technology (Fig. 1) combines small RNA microarray with RNA immunoprecipitation (RIP) to simultaneously measure the modified and unmodified small RNA levels on the same array, providing the vital information to study regulatory impacts of the modification in small RNAs, including miRNA, pre-miRNA, and tsRNA (tRF and tiRNA) classes.

500-Techniques_to_detect_and_quantify_post-transcriptional_modified_small_RNA_expression_levels_v2

Figure 1. Arraystar small RNA modification profiling technology to identify and quantify small RNA post-transcriptional modifications, O8G, m7G, m6A, pseudouridine, and m5C respectively. Modified small RNAs are enriched by immunoprecipitation using a specific antibody, and then identified and quantified by using Arraystar small RNA modification microarray.

 

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METTL3/IGF2BP3 axis inhibits tumor immune surveillance by upregulating N6-methyladenosine modification of PD-L1 mRNA in breast cancer. Wan W, et al. Molecular Cancer, 2022​

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