New Insights into U1 Snrnp Functions in Plant Splicing Mechanisms Using Arabidopsis

By Dr. Chinta SidharthanReviewed by Lily Ramsey, LLMOct 2 2024

The spliceosome in eukaryotes removes non-coding intronic sequences and ligates exons to produce functional messenger ribonucleic acids (mRNA) that code for proteins.

However, while numerous studies have been conducted in metazoans to understand the functions and interactions of the five small nuclear ribonucleoproteins (snRNP) rich in uridine (U) that constitute the spliceosome, the functions and interactions of U1 snRNP in plants remain largely unknown.

In a recent study published in Nature Plants, a team of researchers described the functions and interactions of the U1 snRNP in the plant species Arabidopsis and discussed the genetic resources generated to further explore the non-canonical functions of the U1 snRNP in plants.

​​​​​​​Study: The Arabidopsis U1 snRNP regulates mRNA 3′-end processing. Image Credit: Proonty/Shutterstock.com

Background

The spliceosome is made up of five uridine-rich snRNPs, U1, U2, U4, U5, and U6. These snRNPs play important roles in identifying the splicing signals in genes, removing introns, and ligating exons to produce protein-coding mRNA. The U1 snRNP recognizes the splice site at the 5’ end, initiating the splicing process.

Arabidopsis is a genus of small flowering plants belonging to the Brassicaceae or mustard family. Species of this genus, such as A. thaliana, have been investigated as model plants for numerous genetic studies.

Research has shown that the core and accessory proteins of U1 snRNP in Arabidopsis are conserved, and mutations impacting these components of the spliceosome result in developmental defects.

Furthermore, the U1snRNP is also known to play non-canonical, non-splicing roles, such as regulating transcription and controlling the length of the RNA, as well as telescripting, which ensures proper RNA processing.

However, these non-canonical functions have largely been explored in animal models, and there is a shortage of genetic tools for exploring them in plant systems.

About the study

In the present study, the researchers used A. thaliana plants grown in controlled temperature and light conditions to explore the function and interactions of the U1 snRNP. For this study, artificial microRNAs, which are small, non-coding RNA molecules that help regulate gene expression, were created targeting specific U1 genes such as U1-C and U1-70K.

These microRNAs were then inserted into plasmids and introduced into Arabidopsis plants through a method called floral dipping. This allowed the modified genetic material to integrate into the plants.

The researchers then isolated the RNA from the plants using standardized RNA extraction kits. The extracted RNA was treated to remove deoxyribonucleic acid (DNA) and then used to synthesize complementary deoxyribonucleic acid (cDNA).

Gene expression levels were measured through quantitative polymerase chain reaction (Q-PCR) using the cDNA.

Furthermore, the researchers used the Illumina Hi-Seq system to perform RNA sequencing and analysis by preparing RNA samples, constructing cDNA libraries, and sequencing them.

The sequencing data was cleaned and aligned with reference genomes to analyze gene expression. Additionally, mass spectrometric methods were used for protein analysis to identify the specific proteins that interact with RNA in the U1 snRNP interactome.

The study used various bioinformatics tools to investigate gene expression and splicing. In a separate experiment, the researchers directly sequenced the total RNA using Nanopore technology, which allows real-time RNA sequencing.

This analysis allowed the researchers to study alternative splicing events and RNA-binding proteins that are crucial for understanding the regulation of gene expression.

Major findings

The study identified 214 proteins that were found to interact with the U1 snRNP, including core components such as U1-70K and U1-A and other splicing factors. Many of these proteins were also found to interact with the U2 snRNP, indicating that they played dynamic roles in the splicing process.

The study also revealed that the U1 snRNP plays various other roles in plants besides splicing, including the processing and transport of RNA and the regulation of transcription.

Furthermore, the U1 snRNP was also found to interact with mRNA cleavage and polyadenylation proteins, which suggested that U1 snRNP, along with the cleavage and polyadenylation factors, formed a higher-order complex.

The interactions between U1 snRNP and the cleavage and polyadenylation proteins were confirmed through co-immunoprecipitation assays.

The knockdown of the U1-C and U1-70K genes using microRNAs in Arabidopsis resulted in significant developmental defects such as abnormal leaf growth and dwarfism.

Additionally, RNA sequencing in the knockdown experiments revealed major changes in gene expression and alterations in splicing patterns, such as exon skipping and intron retention. These results highlight the role of U1 snRNP in maintaining the integrity of the transcriptome.

The study also found that U1 snRNP plays two major roles in the regulation of mRNA cleavage and polyadenylation.

The U1 snRNP prevents premature cleavage and polyadenylation within the introns to maintain the integrity of the transcript. Additionally, the U1 snRNP also promotes the use of the proximal cleavage and polyadenylation sites in the 3’ untranslated regions rather than the distal ones, increasing the length of the transcripts.

Conclusions

In summary, the study found that the U1 snRNP in Arabidopsis interacts with a complex network of proteins that are essential for the splicing of the RNA and other functions of the spliceosome.

Mutations in the U1 snRNP genes resulted in developmental defects and major changes in gene expression and splicing patterns.

The study also found that the U1 snRNP also plays two major non-canonical roles involving the mRNA cleavage and polyadenylation proteins that help maintain the integrity and length of the transcripts.