<i>Saccharomyces cerevisiae</i> Ecm2 modulates the catalytic steps of pre-mRNA splicing

Feltz, Clarisse van der; Nikolai, Brandon; Schneider, Charles; Paulson, Joshua C.; Fu, Xingyang; Hoskins, Aaron A.

doi:10.1261/rna.077727.120

Cited by 14 publications

(8 citation statements)

References 58 publications

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“…RNA was isolated using the protocol above. IR700 dye conjugated probes (Integrated DNA Technologies) were used for primer extension with SuperScript III (Thermo Fisher) of the ACT1-CUP1 reporters (2 pmol yAC6: /5IRD700/GGCACTCATGACCTTC) and U6 snRNA (0.4 pmol yU6: /5IRD700/GAACTGCTGATCATGTCTG) (Carrocci et al, 2017; van der Feltz et al, 2021). Primer extension products were visualized on a 7% (w/v) denaturing polyacrylamide gel run at 35 W for 80 min at RT.…”

Section: Methodsmentioning

confidence: 99%

Control of 3ʹ splice site selection by the yeast splicing factor Fyv6

Senn,

Lipinski,

Zeps

et al. 2024

Preprint

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Pre-mRNA splicing is catalyzed in two steps: 5′ splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (1st and 2nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2nd step factor in S. cerevisiae; however, we did not determine how widespread Fyv6's impact is on the transcriptome. To answer this question, we have used RNA-Seq to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3′ SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-EM structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only 2nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of Prp22-dependent, BP distal 3′ SS.

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Section: Methodsmentioning

confidence: 99%

Control of 3ʹ splice site selection by the yeast splicing factor Fyv6

Senn,

Lipinski,

Zeps

et al. 2024

Preprint

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“…Total RNA was isolated from yeast and contaminating DNA was depleted using the MasterPure Yeast RNA Purification Kit (Epicentre, Madison, WI) protocol with minor changes as previously described (Carrocci et al, 2017). IR700 dye conjugated probes (Integrated DNA Technologies, Skokie, IL) were used for primer extension of the ACT1-CUP1 reporter (10 pmol yAC6: /5IRD700/GGCACTCATGACCTTC) and U6 snRNA (2 pmol yU6: /5IRD700/GAACTGCTGATCATGTCTG) (Carrocci et al, 2017; van der Feltz et al, 2021). Primer extension products were visualized on a 7% (w/v) denaturing polyacrylamide gel (42 cm x 22 cm x 0.75 mm) run at 35W for 80 min at RT.…”

Section: Methodsmentioning

confidence: 99%

Biochemical and Genetic Evidence Supports Fyv6 as a Second-Step Splicing Factor inSaccharomyces cerevisiae

Lipinski¹,

Senn²,

Zeps³

et al. 2023

Preprint

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Precursor mRNA (pre-mRNA) splicing is an essential process for gene expression in eukaryotes catalyzed by the spliceosome in two transesterification steps. The spliceosome is a large, highly dynamic complex composed of 5 small nuclear RNAs and dozens of proteins, some of which are needed throughout the splicing reaction while others only act during specific stages. The human protein FAM192A was recently proposed to be a splicing factor that functions during the second transesterification step, exon ligation, based on analysis of cryo-electron microscopy (cryo-EM) density. It was also proposed that Fyv6 might be the functional S. cerevisiae homolog of FAM192A; however, no biochemical or genetic data has been reported to support this hypothesis. Herein, we show that Fyv6 is a splicing factor and acts during exon ligation. Deletion of FYV6 results in genetic interactions with the essential splicing factors Prp8, Prp16, and Prp22; decreases splicing in vivo of reporter gene harboring intron substitutions that limit the rate of exon ligation; and changes 3' splice site (SS) selection. Together, these data suggest that Fyv6 is a component of the spliceosome and the potential functional and structural homolog of human FAM192A.

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“…A very elegant hypothesis is that the RRM of RBM22 would transiently contact the U2 helix IIb, maintaining it in an opened conformation. This would allow it contacting the intron before disruption of the interaction with U2 and wrapping around the intron [156]. Surprisingly though, while the RRM of the yeast Cwc2 is included in a single folding unit located over the region of the U6-ISL and the pre-mRNA, downstream of the 5′SS [141], it is excluded from that region in the human spliceosome, and is located in a more downstream region of the intron [29,98,153].…”

Section: Rbm22 Is Essential For Gene Expression Through Transcription...mentioning

confidence: 99%

“…Finally, it is interesting to note the reflection of Hoskins' team, which states that RBM22 more accurately mimics Ecm2 instead of Cwc2, in terms of interaction with the U6-snRNA. Thus, they believe Ecm2 is a closer structural homolog of RBM22 [156]. However, the evolution of RBM22 has never been truly studied.…”

Section: Rbm22 Is Essential For Gene Expression Through Transcription...mentioning

confidence: 99%

RBM22, a Key Player of Pre-mRNA Splicing and Gene Expression Regulation, Is Altered in Cancer

Soubise

Jiang

Douet‐Guilbert

et al. 2022

Cancers

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RNA-Binding Proteins (RBP) are very diverse and cover a large number of functions in the cells. This review focuses on RBM22, a gene encoding an RBP and belonging to the RNA-Binding Motif (RBM) family of genes. RBM22 presents a Zinc Finger like and a Zinc Finger domain, an RNA-Recognition Motif (RRM), and a Proline-Rich domain with a general structure suggesting a fusion of two yeast genes during evolution: Cwc2 and Ecm2. RBM22 is mainly involved in pre-mRNA splicing, playing the essential role of maintaining the conformation of the catalytic core of the spliceosome and acting as a bridge between the catalytic core and other essential protein components of the spliceosome. RBM22 is also involved in gene regulation, and is able to bind DNA, acting as a bona fide transcription factor on a large number of target genes. Undoubtedly due to its wide scope in the regulation of gene expression, RBM22 has been associated with several pathologies and, notably, with the aggressiveness of cancer cells and with the phenotype of a myelodysplastic syndrome. Mutations, enforced expression level, and haploinsufficiency of RBM22 gene are observed in those diseases. RBM22 could represent a potential therapeutic target in specific diseases, and, notably, in cancer.

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Saccharomyces cerevisiae Ecm2 modulates the catalytic steps of pre-mRNA splicing

Cited by 14 publications

References 58 publications

Control of 3ʹ splice site selection by the yeast splicing factor Fyv6

Control of 3ʹ splice site selection by the yeast splicing factor Fyv6

Biochemical and Genetic Evidence Supports Fyv6 as a Second-Step Splicing Factor inSaccharomyces cerevisiae

RBM22, a Key Player of Pre-mRNA Splicing and Gene Expression Regulation, Is Altered in Cancer

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