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The pathway from gene activation in the nucleus to mRNA translation and decay at specific locations in the cytoplasm is both streamlined and highly interconnected. This review discusses how pre-mRNA processing, including 5' cap addition, splicing, and polyadenylation, contributes to both the efficiency and fidelity of gene expression. The connections of pre-mRNA processing to upstream events in transcription and downstream events, including translation and mRNA decay, are elaborate, extensive, and remarkably interwoven.

Cells control alternative splicing by modulating assembly of the pre-mRNA splicing machinery at competing splice sites. Therefore, a working knowledge of spliceosome assembly is essential for understanding how alternative splice site choices are achieved. In this chapter, we review spliceosome assembly with particular emphasis on the known steps and factors subject to regulation during alternative splice site selection in mammalian cells. We also review recent advances regarding similarities and differences between the in vivo and in vitro assembly pathways, as well as proofreading mechanisms contributing to the fidelity of splice site selection.

Recent work indicates that the posttranscriptional control of eukaryotic gene expression is much more elaborate and extensive than previously thought, with essentially every step of messenger RNA (mRNA) metabolism being subject to regulation in an mRNA-specific manner. Thus, a comprehensive understanding of eukaryotic gene expression requires an appreciation for how the lives of mRNAs are influenced by a wide array of diverse regulatory mechanisms.

The U1 small nuclear ribonucleoprotein particle U1C protein has a zinc finger-like structure (C2H2 motif) at its N terminus, which is conserved from yeast to humans. Mutations of amino acid L13 within this domain rescue the essential function of the helicase protein Prp28p. Prp28p has been implicated in unwinding the 5' splice site (5'ss)-U1 small nuclear RNA (snRNA) base-pairing, to allow replacement of U1 snRNA with U6 snRNA during spliceosome assembly. The L13 phenotype has therefore been interpreted to indicate that WT U1C contributes to 5'ss-U1 snRNA stabilization by binding to the RNA duplex. We show here that an L13 mutant extract cannot form stable base-pairing at room temperature but is permissive for U1-5'ss base-pairing at low temperature. This phenotype is similar to that of a U1C-depleted extract, indicating that the U1C L13 mutation is a strong loss-of-function mutation. The two mutant extracts are unlike a WT extract, which undergoes stable pairing at room temperature but little or no pairing at low temperature. Taken together with previous results and the failure to observe a direct interaction of U1C with the U1-5'ss duplex, the data suggest that U1C contributes indirectly to stable U1-5'ss base-pairing under permissive conditions. A model is proposed to account for the L13 results.

Although it has been known since the late 1970s that intron-containing and intronless versions of otherwise identical genes can exhibit dramatically different expression profiles, the underlying molecular mechanisms have only lately come to light. This review summarizes recent progress in our understanding of how introns and the act of their removal by the spliceosome can influence and enhance almost every step of mRNA metabolism. A rudimentary understanding of these effects can prove invaluable to researchers interested in optimizing transgene expression in eukaryotic systems.

In mammalian cells, significantly more RNA is turned over in the nucleus than in the cytoplasm. However, only recently have we begun to understand the mechanisms and regulation of nuclear RNA decay.