RNA processing plays a pivotal role in the diversification of high eukaryotes transcriptome and proteome. The expression of gene products controlling a variety of cellular and physiological processes depends largely on a complex maturation process undergone by pre-mRNAs to become translation-competent mRNAs. Here we review the different mechanisms involved in the pre-mRNA processing and disclose their impact in the gene regulation process in eukaryotic cells. We describe some viral strategies targeting pre-mRNA processing to control gene expression and host immune response and discuss their relevance as tools for a better understanding of cell biology. Finally, we highlight accumulating evidences toward the occurrence of a translation event coupled to mRNA biogenesis in the nuclear compartment and argue how this is relevant for the production of antigenic peptide substrates for the major histocompatibility complex class I pathway.