1-1 Spliceosomal Small Nuclear RNA Modifications Might Affect Precursor Messenger RNA Splicing

CreativeBiolabs is a contract research organization specializing in mRNA studies. They produce podcast series on mRNA technology and invited David to discuss precursor-messenger RNA splicing and protein synthesis. Splicing involves the removal of non-coding sequences, while protein synthesis is guided by ribosomes. Both processes require post-transcriptional modifications of RNA, such as 2'-oxomethylation and pseudouracil. The function of these modifications in splicing has been explored, and it was found that modified nucleotides play a role in small nuclear RNA assembly and precursor-messenger RNA splicing. Recombinant systems have been developed to study these modifications, and experiments have shown their importance in splicing. The lack of modified small nuclear RNA affects splicing efficiency. Further studies have confirmed the requirement of modifications for small nuclear RNA assembly and precursor-messenger RNA splicing. Welcome to CreativeBiolabs Science Channel. CreativeBiolabs is a specialized contract research organization supporting mRNA studies with all-round solutions covering mRNA synthesis, modification, and mRNA therapeutics development. With an unwavering pursuit of innovation and lifelong learning, we keep on producing podcast series related to mRNA technology based on our knowledge and practical experience gained through years of exploration in this area. Subscribe to our channel and keep updated with our podcasts. Good evening, dear friends. Thank you for tuning in to CreativeBiolabs podcast series. Today, we invited David to our program. Thanks for joining us, David. Thanks for inviting me, Connie. It's great to be here. It is well known that the accuracy of genetic information flow from DNA to protein or gene expression is crucial to the survival of organisms. The processing of precursor-messenger RNA and the synthesis of protein are inherent in the gene expression of higher eukaryotes. In other words, precursor-messenger RNA splicing and protein translation are the two main steps of eukaryotic gene expression. They need to produce precise gene products. David, can you give us a brief introduction on these two processes? Sure. Precursor-messenger RNA splicing is a kind of RNA processing reaction. Two successive transesterification reactions lead to the removal of non-coding sequence, intron, coding sequence, and exon before the production of mature messenger RNA, which can then be exported to the cytoplasm where it directs the synthesis of protein. Both precursor-messenger RNA splicing and protein synthesis are multi-step processes coordinated by large and dynamic ribonucleoprotein complexes. What components are needed for the splicing of precursor RNAs? In the case of the spliceosome, the machinery responsible for intron removal, five uridyl-rich small nuclear RNAs, namely U1, U2, U4, U5, and U6, as well as a number of proteins, orchestrate the splicing of precursor-messenger RNAs. And what about for protein synthesis? In higher eukaryotes, the ribosome is the mechanism guiding protein synthesis. It is composed of four ribosomal RNAs, namely 5s, 5.8s, 18s, 28s, and many protein factors. Interestingly, both small nuclear RNA and ribosomal RNA of spliceosomes contain a variety of post-transcriptional modified nucleotides. That's right. RNA components contain a large number of post-transcriptional modifications, including 2'-oxomethylation and pseudouracil. They are important functionally. So far, the two most abundant modifications are 2'-oxomethylation and pseudouracil. 2'-oxomethylation is a kind of RNA skeleton targeting reaction. It leads to the introduction of methyl at the 2'-oxygen position of the sugar ring. Pseudouridine is a specific modification of uridine, resulting in the five ribosyl isomers of uridine. So in the past decades, people have been trying to clarify the mechanism of these modifications and their molecular functions. What progress has been made? So far, the most significant progress has been made in introducing changes. In recent years, especially in the last 10 years, the field of RNA modification has made great progress and accumulated a lot of data. But in contrast, the knowledge about the function of post-transcriptional modified nucleotides went relatively backward. This is partly due to the lack of appropriate analytical and experimental systems. Let's move on to the functional aspects of spliceosomal small nuclear RNA modifications, so that we can better understand how post-transcriptional modifications affect gene expression. What can you tell us about the modification of spliceosomal small nuclear RNAs? Before 1951, RNA was thought to be composed of four classical ribonucleosides, namely adenosine, guanosine, uridine, and cytidine. And the turning point is really at 1951, when the field of RNA modification was born. At that time, an unknown nucleoside was reported and identified as 5'-ribosylurosyl. It was found in the RNA hydrolysate of the calf liver. Soon after, other modified nucleosides were discovered, including various 2'-oxomethylribose derivatives. The spliceosomal small nuclear RNAs were initially identified as small nuclear RNAs rich in uracil. However, it is surprising that when the exact nucleotide composition is finally determined, it is clear that these RNAs contain some pseudouridine and two oxomethylated residues. Whether these modified nucleotides play an important role in precursor-messenger RNA splicing has become a central issue. Any clue that spliceosomal small nuclear RNA modifications might affect precursor-messenger RNA splicing? It is a long story, but we'll make it short. Although spliceosomal small nuclear RNA has been extensively post-transcriptionally modified since it was first discovered in the 1960s and 1970s, it was not until the early 1990s that the functional role of these modifications was explored. Around 1988, scientists did notice the fact that the modified nucleotides were not randomly distributed throughout the small nuclear RNA. In fact, modifications are particularly concentrated in regions of functional significance, such as those involved in RNA and RNA interactions. In addition, the analysis of the distribution of modified nucleotides in spliceosomal small nuclear RNA of different organisms showed that the modified sites were conserved throughout the evolution. When did scientists begin to evaluate the function of spliceosomal small nuclear RNA? In the early 1990s, a number of functional reconstruction systems were developed to directly evaluate the function of spliceosomal small nuclear RNA and its modification in spliceosomal small nuclear RNA splicing. In particular, a scientist developed an in-vitro youth small nuclear RNA assembly and modification system from breast cancer HeLa cells and nuclear extracts. This system not only can dissociate youth small nuclear RNA, but it can also dissociate functional youth small nuclear RNP. What did he find? To investigate the effect of 5-fluorouracil incorporation on small nuclear RNP assembly, he demonstrated that the presence of 5-fluorouracil in youth 2 small nuclear RNA prevented the formation of salt-tolerant complexes. It was analyzed by cesium sulfate buoyancy density gradient centrifugation. This data, together with previous data show the inhibitory effect of pseudouracil acidification on small nuclear RNA containing 5-fluorouracil, suggesting the role of pseudouracil acidification in the biogenesis of youth 2 small nuclear RNP. How can we distinguish whether the modification of precursor messenger RNA is accomplished by spliceosomal small nuclear RNAs in vivo or by spliceosomal small nuclear RNAs introduced in vitro? Recombinant systems have been developed, including specifically depleting one of the endogenous spliceosomal small nuclear RNA and then supplementing the corresponding snRNA synthesized in vitro. Since the small nuclear RNA synthesized in vitro is not modified, the ability or lack of ability of precursor messenger RNA splicing before RNA reconstruction will indicate whether a modification is needed for precursor messenger RNA splicing. Surprisingly, U1, U4 and U6 synthesized in vitro have been shown to be effective in reconstituting precursor messenger RNA splicing in mammalian extracts that deplete their respective endogenous small nuclear RNA. Can you give us an example that small nuclear RNA synthesized in vitro can participate in the splicing of precursor messenger RNA? Sure. The U6 small nuclear RNA synthesized in vitro is one. It successfully saved the precursor messenger RNA splicing in the depleted cell-free extract of U6 from Ascaris embryo. There are also the U2 and U6 synthesized in vitro. They both can restore the splicing of precursor messenger RNA in yeast cell extracts. These initial data pose a rather thorny problem because the lack of U small nuclear RNP assembly may have a negative impact on precursor messenger RNA splicing. Has any of the recombinant experiments you mentioned above detected the modification of small nuclear RNA that has been transcribed in vitro? Right. Not yet. In fact, U2 small nuclear RNA transcribed in vitro can be easily added to yeast splicing extract for modification. Therefore, whether modified nucleotides contribute to precursor messenger RNA splicing remains an open question. Through what experiments can we prove that the modified nucleotides in U2 small nuclear RNA indeed play a role in splicing? As early as in 1995, a research group studied the function of spliceosomal small nuclear RNA in HeLa reconstruction system. Interestingly, although U2 small nuclear RNA synthesized in vitro could not reconstruct precursor messenger RNA splicing in U2 small nuclear RNA-deficient extracts, cell-derived U2 small nuclear RNA successfully restored splicing. This suggests the role of the modified nucleotides in U2 small nuclear RNA in splicing. There are also some further analyses confirming that there was not pseudobreak of U2 small nuclear RNA in the extract. Is there any definitive evidence that spliceosomal small nuclear RNA modifications are required for small nuclear RNP assembly and precursor messenger RNA splicing? I know some studies have been carried out, like the biogenesis and splice assembly of U2 small nuclear RNP in Xenopus oocytes were studied. And they did detect the precursor messenger RNA splicing and splice assembly. Interestingly, unmodified or low-modified U2 small nuclear RNA could not support splicing. A detailed analysis showed that the U2 small nuclear RNA could not participate in the assembly of any high-order splicing complex. How do we detect the assembly of U2 small nuclear RNP? We can use anti-SNRNP immunoprecipitation and glycerin gradient precipitation. Some experimental data has shown that the lack of modified U2 small nuclear RNA can only form 12s small nuclear RNP, and no mature form of U2 small nuclear RNP is observed. These results suggest that the transition from 12s small nuclear RNP to functional 17s small nuclear RNP requires U2 small nuclear RNA modification. What is the main form of U2 small nuclear RNA synthesized in vitro? Interestingly, the main form is the 17s particle so that the ability to support splicing can be regained. The analysis of U2 small nuclear RNA changes with time showed that these RNAs could be effectively modified after long-term incubation in Xenopus oocyte reconstruction system. Therefore, there is a good correlation between the modification status of U2 small nuclear RNA and its ability to participate in precursor messenger RNA splicing. Talking about the precursor messenger RNA splicing, which site modification is necessary for it to happen? A team constructed a chimeric U2 small nuclear RNA in which none of the regions of the U2 small nuclear RNA contained modification. This experiment proved that the modification in 275-foot most of the nucleotides is necessary for the splicing of precursor messenger RNA. The functional importance of these modified nucleotides was later confirmed. Recently, another team used U2 small nuclear RNA containing 5-fluorouracil, a U2-specific pseudouracil dehydrogenase inhibitor, to further analyze the importance of U2 small nuclear RNA modification. The results showed that effective precursor messenger RNA splicing and splice assembly in Xenopus oocytes required pseudouracil in the branch site recognition region. I have seen reports that Saccharomyces cerevisiae precursor messenger RNA splicing mechanism is one of the most widely studied splicing assembly and precursor messenger RNA splicing catalytic systems. Can you introduce us to the genetic anatomy of small nuclear RNA modification in Saccharomyces cerevisiae? Saccharomyces cerevisiae is an ideal organism to analyze the function of post-transcriptional modified nucleotides in spliceosomal small nuclear RNA. Surprisingly, so far, number 2-omethylation has been observed in Saccharomyces cerevisiae spliceosomal small nuclear RNA. But later, 6 pseudouridines were identified, 5 of which were conserved in mammals. There are 2 in U1, 3 in U2, and 1 in U5. The genes encoding U2 small nuclear RNA pseudouricase have been identified. Does pseudo-deoxyribonucleic acid in yeast spliceosomal small nuclear RNA have a synergistic effect? Good question, but the answer is we don't know yet. We still need a detailed analysis of small nuclear RNA pseudostaining of other yeast spliceosomes. However, the fact that they are conserved throughout evolution suggests their function. Okay, thanks David for sharing your expertise with us today. Thanks, everyone, for listening. We will continue our discussion on RNA modifications and gene expressions next week. See you next time!