An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8

Price, Argenta; Görnemann, Janina; Guthrie, Christine; Brow, David A.

doi:10.1261/rna.041970.113

Cited by 19 publications

(25 citation statements)

References 66 publications

(113 reference statements)

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“…We suggest that Prp45 is important for timely (i.e., cotranscriptional) U2 snRNP association as well as the subsequent stages of spliceosome formation. The effect of the PRP45 allele on U2 resembles the "early roles" reported for PRP8 and PRP28 (Price et al 2014) in that it occurs before the stable association of the respective splicing factor with pre-mRNA (Ohi et al 2002).…”

Section: Introductionsupporting

confidence: 60%

Nineteen complex–related factor Prp45 is required for the early stages of cotranscriptional spliceosome assembly

Hálová

Gahura

Převorovský

et al. 2017

RNA

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Splicing in has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in (1-169) cells. We also measured pre-mRNA accumulation of the meiotic gene, which depends on the expression of Mer1 factor for splicing. (1-169) cells accumulated approximately threefold higher levels of pre-mRNA than WT cells only when splicing was induced. To monitor cotranscriptional splicing, we determined the presence of early spliceosome assembly factors and snRNP complexes along the and genes. We found that (1-169) hampered the cotranscriptional recruitment of U2 and, to a larger extent, U5 and NTC, while the U1 profile was unaffected. The recruitment of Prp45(1-169) was impaired similarly to U5 snRNP and NTC. Our results imply that Prp45 is required for timely formation of complex A, prior to stable physical association of U5/NTC with the emerging pre-mRNA substrate. We suggest that Prp45 facilitates conformational rearrangements and/or contacts that couple U1 snRNP-recognition to downstream assembly events.

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Section: Introductionsupporting

confidence: 60%

Nineteen complex–related factor Prp45 is required for the early stages of cotranscriptional spliceosome assembly

Hálová

Gahura

Převorovský

et al. 2017

RNA

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“…At this stage, U1 snRNA is displaced from the pre-mRNA 5′-SS, a process that requires the activity of the Prp28 protein. 12,13 Concomitantly, the U4/U6 base pairing interaction is disrupted by the Brr2 enzyme, [14][15][16] and U4 snRNA, as well as all U4/U6-associated proteins, are displaced from the spliceosome. U4/U6 disruption allows U6 snRNA to engage Conserved RNA-binding elements in the tunnel of the N-terminal cassette, brown; C-terminal cassette, light brown; hPrp8 Jab1 , gold with semi-transparent surface.…”

Section: Pre-mrna Splicing By the Spliceosomementioning

confidence: 99%

Novel regulatory principles of the spliceosomal Brr2 RNA helicase and links to retinal disease in humans

et al. 2014

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For each round of pre-mRNA splicing, a spliceosome is assembled anew on its substrate. RNA-protein remodeling events required for spliceosome assembly, splicing catalysis, and spliceosome disassembly are driven and controlled by a conserved group of ATPases/RNA helicases. The activities of most of these enzymes are timed by their recruitment to the spliceosome. The Brr2 enzyme, however, which mediates spliceosome catalytic activation, is a stable subunit of the spliceosome, and thus, requires special regulation. Recent structural and functional studies have revealed diverse mechanisms whereby an RNaseH-like and a Jab1/MPN-like domain of the Prp8 protein regulate Brr2 activity during splicing both positively and negatively. Reversible Brr2 inhibition might in part be achieved via an intrinsically unstructured element of the Prp8 Jab1/MPN domain, a concept widespread in biological systems. Mutations leading to changes in the Prp8 Jab1/MPN domain, which are linked to a severe form of retinitis pigmentosa, disrupt Jab1/MPN-mediated regulation of Brr2.

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“…2), indicating reduced and possibly delayed U2 recruitment, which agrees with our previous observation of Prp45 depletion causing a first step of splicing defect. The elevated U1 signal is likely caused by failure to form B complex, which normally involves U1 release [37]. In the absence of either Prp16 or Prp22, we again observe lower occupancy of Lea1 (U2) and higher occupancy of Prp40 (U1), similar to depletion of Prp45.…”

Section: Depletion Of Prp16 or Prp22 Causes A First Step Of Splicing mentioning

confidence: 51%

Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo

et al. 2019

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Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this ‘feed-back’ effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.

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An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8

Cited by 19 publications

References 66 publications

Nineteen complex–related factor Prp45 is required for the early stages of cotranscriptional spliceosome assembly

Nineteen complex–related factor Prp45 is required for the early stages of cotranscriptional spliceosome assembly

Novel regulatory principles of the spliceosomal Brr2 RNA helicase and links to retinal disease in humans

Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo

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