Precursor messenger RNA (pre-mRNA) splicing is an essential step in the flow of information from DNA to protein in all eukaryotes Research over the past four decades has molecularly delineated the splicing pathway, including characterization of the detailed splicing reaction, definition of the spliceosome and identification of its components, and biochemical analysis of the various splicing complexes and their regulation Structural information is central to mechanistic understanding of pre-mRNA splicing by the spliceosome X-ray crystallography of the spliceosomal components and subcomplexes is complemented by electron microscopy of the intact spliceosome In this Review, I discuss recent atomic-resolution structures of the intact spliceosome at different stages of the splicing cycle These structures have provided considerable mechanistic insight into pre-mRNA splicing and have corroborated and explained a large body of genetic and biochemical data Together, the structural data have proved that the spliceosome is a protein-directed metalloribozyme

Mechanistic insights into precursor messenger RNA splicing by the spliceosome

mRNA maturation in trypanosomes differs from the process in most eukaryotes mainly because protein-coding genes are transcribed into polycistronic RNAs in this organism ([78][1]). Studies from the ongoing genome project suggest that the entire chromosome may be transcribed as large transcripts, but

https://ec.asm.org/content/eukcell/2/5/830.full.pdf

trans and cis Splicing in Trypanosomatids: Mechanism, Factors, and Regulation

https://www.cell.com/current-biology/pdf/S0960-9822(07)00345-4.pdf

RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2

The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing. It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated. The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome. The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here.

Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery

RNA helicases represent a large family of proteins that have been detected in almost all biological systems where RNA plays a central role. They are ubiquitously distributed over a wide range of organisms and are involved in nuclear and mitochondrial splicing processes, RNA editing, rRNA processing, translation initiation, nuclear mRNA export, and mRNA degradation. RNA helicases are described as essential factors in cell development and differentiation, and some of them play a role in transcription and replication of viral single-stranded RNA genomes. Comparisons of the conserved sequences reveal a close relationship between them and suggest that these proteins might be derived from a common ancestor. Biochemical studies have revealed a strong dependence of the unwinding activity on ATP hydrolysis. Although RNA helicase activity has only been demonstrated for a few examples yet, it is generally believed that all members of the largest subgroups, the DEAD and DEAH box proteins, exhibit this activity.

The protein family of RNA helicases.

To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1994.tb06331.x

A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.

The RNA helicase-like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of interactions of PRP2 with spliceosomal components, PRP2 protein was stalled in splicing complexes by two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre-mRNAs, as demonstrated by UV-crosslinking experiments. The use of various mutant and truncated pre-mRNAs revealed that this interaction requires a spliceable pre-mRNA and an assembled spliceosome; a 3' splice site is not required. To extend these observations to the wild-type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre-mRNA in these spliceosomes in an ATP-independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S-ATP showed that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre-mRNA. As PRP2 is an RNA-stimulated ATPase, these experiments strongly suggest that the pre-mRNA is the native co-factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, we propose that the pre-mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1994.tb06332.x

The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.

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A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.

The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.