Pseudouridine in RNA

RNA not only acts as an intermediate or effector molecule in protein synthesis, but also has a direct functional effect on gene expression. Therefore, the importance of RNA modification has become increasingly prominent. Many RNAs in nature can be post-transcriptionally modified. This modification not only has important functions, such as enhancing the degradation of RNA, promoting or inhibiting the processing and formation of RNA, changing the activity of RNA or as a quality control mechanism of RNA, but also related to the pathogenesis of human diseases including cancer. Currently, scientists have discovered more than 100 different types of RNA modifications. The four bases of RNA and ribose can be modified targets. Among them, pseudouridine modification is the earliest and most abundant RNA modification. Pseudouridine is also known as the “fifth nucleotide” in RNA. Pseudouridine is the 5-ribosyl isomer of uridine, in which ribosyl is attached to a carbon instead of a nitrogen atom. Pseudouridine is widely present in various RNA species (tRNA, rRNA, snRNA, and snoRNA).

The chemical structure of uridine and pseudouridine

Formation of pseudouridine

Pseudouridine is produced by the isomerization of uridine catalyzed by pseudouridine synthase. There are two main mechanisms for the formation of pseudouridine in RNA.

One is a mechanism that only relies on pseudouridine synthase. In this mechanism, pseudouridine synthase is RNA-independent, and it plays a role of recognition and catalysis at the same time. Different pseudouridine synthase has specificity for the corresponding substrate RNA. Through the combination of biochemical and bioinformatics methods, pseudouridine synthase is mainly divided into five protein families: TruA, TruB, TruD, RluA and RsuA. For example, the pseudouridine synthase of the TruA ​​family catalyzes the isomerization of uridine on the anticodon loop of tRNA. The pseudouridine synthase of the RsuA family mainly catalyzes the pseudouridine modification on rRNA in prokaryotes.

The other is a mechanism that relies on the box H/ACA ribonucleoprotein. In this mechanism, pseudouridine synthase is RNA-dependent. RNA plays a recognition role, and the pseudouridine synthase bound to it plays a catalytic role. Box H/ACA ribonucleoprotein is composed of H/ACA RNA and pseudouridine synthase. Box H/ACA RNA contains a loop complementary to the sequence of the substrate RNA, which in turn determines the specificity of the pseudouridine modification site. Therefore, H/ACA RNA plays a major role in recognition in the catalytic process. In the box H/ACA ribonucleoprotein, pseudouridine synthase, such as Cbf5, can form the isomerization of uridine. Box H/ACA ribonucleoprotein mainly catalyzes the production of pseudouridine modification on rRNA and snRNA.

Only the first mechanism exists in prokaryotes, and both mechanisms exist in eukaryotes.

Function of pseudouridine modification in RNA

  • In rRNAs

Pseudouridine modifications in rRNA are ubiquitous in the small subunit (SSU) and large subunit (LSU) of rRNA, and are mainly distributed in functionally important structural regions, such as peptidyltransferase center (PTC), decoding center and A-site finger (ASF) region. At present, inferences about the function of pseudouridine in rRNA are largely derived from the known biochemical and biophysical effects of pseudouridine residues in other cases and our inferences about pseudouridine in a single rRNA species Understanding of the distribution. The distribution of pseudouridine implies that pseudouridine modification can affect the function of rRNA. Pseudouridine modification in these regions may affect rRNA folding, ribosome assembly, and maintenance of the corresponding high-level structure.

The deletion of all snoRNA related to pseudouridine in the PTC region in yeast cells will have a greater impact on the growth rate. The deletion of pseudouridine in the decoding center will slow down the growth rate, and the small subunit RNA has obvious defects.

  • In Small Nuclear and Nucleolar RNAs

In eukaryotes, pseudouridine is present in the major spliceosome snRNA (U1, U2, U4, U5, and U6) and the minor vertebrate variants responsible for splicing the AU/AC intron (U12, U4atac, and U6atac). Pseudouridine modification has basically occurred in all snRNAs. These modifications are highly conserved among different species, but they also have specific variations in organisms or taxonomy. Similar to rRNA, pseudouridine in snRNA is also mainly concentrated in functionally important areas. For example, the modified distribution area of ​​pseudouridine in U1 snRNA plays an important role in identifying the 5’splice site. Studies have shown that the modified distribution area of ​​pseudouridine in snRNA is involved in the interaction between RNA and RNA or between RNA and protein. These interactions play a role in the shearing process and are related to the assembly and function of the spliceosome.  

Pseudouridine modifications have also been found in snoRNA, especially U3, U8, snR4 and snR8, but the formation or function of pseudouridine modifications in snoRNA have not been studied in detail.

  • In tRNAs

Pseudouridine is present in almost all tRNAs. Pseudouridine on tRNA is mainly distributed in the universal TΨC stem loop. In archaebacteria, eubacteria and eukaryotes, as well as mitochondria and chloroplasts, pseudouridine on tRNA is also distributed in the D stem and the anticodon stem and loop. Pseudouridine on tRNA can affect the local structure of the domain where it is distributed, thereby affecting decoding activity, improving the accuracy of translation, and helping to maintain a proper reading frame. For example, the deletion of pseudouridine in the ASL region of the transfer RNA in Escherichia coli and Salmonella typhimurium resulted in abnormal transcription and increased doubling time.

  • In mRNAs

Pseudouridine modifications are mostly found in rRNA, tRNA and snRNA. Recently, researchers discovered that messenger RNA pseudouridine is also a widespread and conservative phenomenon. Because RNA secondary structure is closely related to many aspects of mRNA metabolism and function, pseudouridine may induce structural changes, thereby affecting function. In addition, pseudouridylation can also introduce post-transcriptional gene coding, thereby diversifying the proteome. The translation level of in vitro transcribed mRNA containing pseudouridine was significantly higher than that of mRNA without pseudouridine modification.

It has been found that pseudouridine modification exists in tRNA, rRNA, snRNA, snoRNA and mRNA, but the formation mechanism and function of pseudouridine modification have not been thoroughly studied. With the further development of future technology, a new field of pseudouridine research will be opened.