Nm/2'-O-Methylation
Nm, or 2'-O-methylation, denotes a pivotal co- or post-transcriptional modification occurring in RNA. This process entails the addition of a methyl group (–CH3) to the 2' hydroxyl (–OH) of the ribose moiety. Widely prevalent across various RNA species, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (snRNA), Nm showcases a high degree of conservation and abundance. Its presence is also noted in messenger RNA (mRNA) and at the 3'-end of small non-coding RNAs (sncRNAs) like microRNAs (miRNAs) and small-interfering RNAs (siRNAs) in plants, flies, and animals.
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Fig 1. 2'-O-methylated ribonucleoside (Nm) (Dimitrova D.G., et al. 2019).
Nm's impact on RNA function is multifaceted. It enhances hydrophobicity, shields against nuclease activity, stabilizes helical structures, and influences interactions with proteins or other RNAs. For instance, Nm augments the thermodynamic stability of RNA base pairs, fortifies A-form RNA duplexes, but also can disrupt RNA tertiary structures and impede RNA-protein interactions.
Methods for Detecting Nm
Detecting Nm modifications has historically posed challenges. Techniques like liquid chromatography coupled with mass spectrometry (LC/MS) and two-dimensional thin-layer chromatography (2D-TLC) are effective but labor-intensive. Other methods exploit Nm's protective effect against cleavage or its ability to impede reverse transcriptase. Despite advancements, methods often trade sensitivity for ease of execution and applicability.
RiboMethseq, a sequencing method based on Nm's protection against alkaline hydrolysis, offers high throughput but is limited to longer RNAs. Recent innovations, like methods detecting Nm at the 3' end of small RNAs, broaden applicability. Despite requiring more starting material, these methods offer advantages in sequencing depth.
Nm in tRNA
Transfer RNA (tRNA) molecules, essential for protein synthesis, undergo extensive modifications, among which Nm stands out as one of the most abundant. The loss of certain Nm modifications can disrupt cellular processes and lead to diseases. Nm's function within tRNAs varies depending on its location, contributing to the overall stability of the tRNA structure and facilitating proper translation.
FTSJ1, also known as MRX9, TRMT7, or JM23, is a well-characterized human tRNA 2'-O-methyltransferase. It targets specific positions within the anticodon loop of tRNAPhe and tRNATrp, belonging to the RRMJ/fibrillarin superfamily of RNA methyltransferases. Its homologs in bacteria and yeast, FTSJ/RRMJ and TRM7 respectively, participate in similar modification circuitries crucial for efficient translation.
Notably, mutations in the FTSJ1 gene have been linked to non-syndromic X-linked intellectual disability (NSXLID). Loss-of-function mutations in FTSJ1 lead to neurodevelopmental disorders, emphasizing the importance of Nm modifications in tRNAs for cognitive processes.
Further studies have explored associations between FTSJ1 and cognitive abilities, revealing potential links between genetic variations in FTSJ1 and cognitive traits in males. However, deeper investigations are warranted to fully establish this connection.
FTSJ1's role extends beyond intellectual disability, with implications in diseases like epilepsy. Similarly, other enzymes involved in tRNA Nm modifications, such as TRMT44, have been implicated in idiopathic epilepsy, underscoring the broad impact of Nm modifications on health.
Nm in rRNAs
Nm modifications in rRNAs, primarily guided by box C/D snoRNAs like SNORD26, SNORD44, and SNORD78, play crucial roles in ribosome biogenesis and translation accuracy. Deficiencies in these snoRNAs, as seen in Prader-Willi syndrome (PWS), can lead to severe developmental defects.
Fibrillarin (FBL), an essential methyltransferase associated with snoRNAs, is highly conserved across species and vital for ribosome production. Its dysregulation is linked to various diseases, including cancer, where it promotes increased ribosome biogenesis and tumor growth. Additionally, autoantibodies against FBL are implicated in autoimmune diseases like systemic sclerosis.
FTSJ2, another rRNA methyltransferase, is overexpressed in lung cancer but exhibits complex roles in tumor progression, potentially being advantageous in early stages but detrimental in later stages.
Nm in mRNAs
Nm modifications in mRNAs, whether occurring co- or post-transcriptionally, exert profound effects on their steady state levels. Initially observed primarily in mRNA caps, recent findings indicate internal modifications within the coding DNA sequence (CDS). The absence of Nm can indirectly impact mRNA expression by influencing small nuclear RNA (snRNA)-mediated mRNA splicing.
The cap structure, crucial for mRNA stability and processing, includes "cap 0" with a methylguanosine addition, followed by "cap 1" and "cap 2" with additional 2'-O-methylations. CMTR1 and CMTR2 are responsible for these modifications, with implications for mRNA stability and immune response regulation.
In asthma, CMTR1 dysregulation correlates with exacerbations, possibly due to its involvement in viral defense mechanisms. In Alzheimer's disease, CMTR1 upregulation suggests a role in the pro-inflammatory environment linked to disease progression. Although not directly implicated in cancer, CMTR1 may play a role in oncogenesis through mechanisms such as alterations in gene expression or fusion oncogenes.
Furthermore, mRNA splicing, facilitated by small nuclear ribonucleoproteins (snRNPs), is crucial for mRNA maturation. Modifications on snRNAs, guided by scaRNAs, impact splicing efficiency and could contribute to diseases like cerebellar ataxia. Additionally, internal Nm modifications in mRNA CDS influence translation fidelity, potentially affecting cellular functions.
Nm in Small RNAs Silencing Pathways
Small non-coding RNA (sncRNA) pathways regulate gene expression through transcriptional or post-transcriptional silencing. These pathways include miRNAs, siRNAs, and piRNAs, which guide Argonaute (Ago) proteins to target mRNAs. Nm modification at the 3'-end of these RNAs, catalyzed by HENMT1, is crucial for stability, especially in the PIWI/piRNA pathway protecting germ cells from transposable elements. In mice, Henmt1 mutation disrupts spermatogenesis, leading to infertility due to piRNA instability. In humans, HENMT1 is expressed in germ cells and implicated in testicular germ cell tumors. Despite its germline role, piRNAs are also found in somatic cells across species, suggesting broader functions. In Drosophila, HENMT1 affects somatic-piRNA processes and neurodegeneration, highlighting its significance beyond germ cells.
In conclusion, Nm modifications play diverse roles in RNA biology, influencing stability, structure, and function across various RNA species. With implications in crucial cellular processes and diseases ranging from neurodevelopmental disorders to cancer, the study of Nm modifications continues to uncover their intricate mechanisms and broad impact on health and disease.
References
- Dimitrova D.G., et al. RNA 2'-O-methylation (Nm) modification in human diseases. Genes. 2019, 10(2): 117.
- Jaafar M., et al. 2' O-Ribose methylation of ribosomal RNAs: natural diversity in living organisms, biological processes, and diseases. Cells. 2021, 10(8): 1948.
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