The presence or absence of certain chemical modifications in RNA can significantly affect pre-mRNA splicing and ultimately impact gene expression in eukaryotic cells. Pre-mRNA splicing is a critical process in the maturation of mRNA, where introns are removed, and exons are joined together to form a mature mRNA molecule that can be translated into a protein. Chemical modifications in RNA can influence this process in several ways:1. Alternative splicing: Chemical modifications can lead to alternative splicing, which is the process by which different combinations of exons are joined together to produce multiple mRNA molecules from a single pre-mRNA molecule. This can result in the production of different protein isoforms with distinct functions. Modifications such as N6-methyladenosine m6A have been shown to affect alternative splicing by influencing the recruitment of splicing factors or by altering the RNA structure.2. Splicing efficiency: Chemical modifications can also affect the efficiency of the splicing process. For example, the presence of m6A near splice sites can promote the recruitment of splicing factors, thereby enhancing splicing efficiency. On the other hand, modifications such as pseudouridine can stabilize RNA secondary structures, which may hinder the access of splicing factors to the splice sites and reduce splicing efficiency.3. Splicing fidelity: Chemical modifications can impact the fidelity of the splicing process, which refers to the accuracy with which introns are removed and exons are joined together. Modifications such as 5-methylcytosine m5C have been shown to affect splicing fidelity by altering the recognition of splice sites by the spliceosome.4. Splicing regulation: Chemical modifications can also play a role in the regulation of splicing in response to cellular signals or environmental cues. For example, reversible phosphorylation of RNA-binding proteins can modulate their interaction with target pre-mRNAs, thereby affecting splicing patterns in response to cellular signaling pathways.5. mRNA stability and translation: Some chemical modifications can also affect the stability and translation efficiency of the mature mRNA molecule. For example, m6A has been shown to influence mRNA stability by recruiting specific RNA-binding proteins that can either promote or inhibit mRNA degradation. Additionally, m6A can also affect translation efficiency by modulating the interaction between mRNA and the translation machinery.In summary, the presence or absence of certain chemical modifications in RNA can have a profound impact on pre-mRNA splicing and gene expression in eukaryotic cells. These modifications can influence alternative splicing, splicing efficiency, splicing fidelity, splicing regulation, and mRNA stability and translation, ultimately affecting the production of functional proteins and cellular processes.