The Splicing ATPase Prp43p Is a Component of Multiple Preribosomal Particles

150

To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5′ exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product.RNA helicase | RNA processing | small nuclear RNA | ribonucleoprotein particle | Saccharomyces cerevisiae I n nuclear pre-mRNA splicing, the excision of introns is catalyzed by the spliceosome, a ribonucleoprotein machine comprising five snRNAs and ∼80 conserved proteins (for reviews, see ref. 1 and references therein). This machine assembles de novo on each pre-mRNA substrate and must rearrange its components throughout the splicing cycle through the activity of at least eight DExD/H box ATPases (2). The protein and RNA components of the spliceosome recognize the conserved elements of the intron at the 5′ splice site, the branch site, and the 3′ splice site. The RNA and possibly protein components also play key roles in catalyzing splicing, which occurs by two transesterification reactions (1). In the first reaction, the branch site adenosine attacks the 5′ splice site, generating a free 5′ exon and a lariat intermediate. In the second reaction, the 5′ exon attacks the 3′ splice site, excising the intron and ligating the exons. To establish specificity in splicing, the spliceosome discriminates optimal from suboptimal substrates.The specific pathway that discriminates against a suboptimal substrate depends on the extent to which a substrate is suboptimal. A grossly suboptimal substrate will fail to bind the spliceosome. Although such pre-mRNAs can be degraded in the nucleus (3), they can also be exported and then degraded in the cytoplasm (4-8). In contrast, optimal substrates are specifically retained in the nucleus to favor splicing (9).A nearly optimal substrate engages the spliceosome, necessitating more sophisticated fidelity ...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Spliceosome discards intermediates via the DEAH box ATPase Prp43p

Mayas

Maita²,

Semlow

et al. 2010

150

“…However, it would presumably not be feasible to dedicate a unique chaperone to each structured RNA, much less to each misfolded conformation of each RNA. Even some DExD͞H-box proteins that were initially identified with a unique substrate have been found subsequently to interact functionally with additional RNAs (5,(54)(55)(56), and there is almost certainly a need for general chaperones that can facilitate folding of multiple RNAs, as demonstrated for CYT-19 and its yeast ortholog MSS116 (21,23,24).…”

Section: Discussionmentioning

confidence: 99%

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

Tijerina

Bhaskaran

Russell

2006

163

We explore the interactions of CYT-19, a DExD͞H-box protein that functions in folding of group I RNAs, with a well characterized misfolded species of the Tetrahymena ribozyme. Consistent with its function, CYT-19 accelerates refolding of the misfolded RNA to its native state. Unexpectedly, CYT-19 performs another reaction much more efficiently; it unwinds the 6-bp P1 duplex formed between the ribozyme and its oligonucleotide substrate. Furthermore, CYT-19 performs this reaction 50-fold more efficiently than it unwinds the same duplex free in solution, suggesting that it forms additional interactions with the ribozyme, most likely using a distinct RNA binding site from the one responsible for unwinding. This site can apparently bind double-stranded RNA, as attachment of a simple duplex adjacent to P1 recapitulates much of the activation provided by the ribozyme. Unwinding the native P1 duplex does not accelerate refolding of the misfolded ribozyme, implying that CYT-19 can disrupt multiple contacts on the RNA, consistent with its function in folding of multiple RNAs. Further experiments showed that the P1 duplex unwinding activity is virtually the same whether the ribozyme is misfolded or native but is abrogated by formation of tertiary contacts between the P1 duplex and the body of the ribozyme. Together these results suggest a mechanism for CYT-19 and other general DExD͞H-box RNA chaperones in which the proteins bind to structured RNAs and efficiently unwind loosely associated duplexes, which biases the proteins to disrupt nonnative base pairs and gives the liberated strands an opportunity to refold.group I RNA ͉ RNA folding ͉ RNA unwinding ͉ Tetrahymena ribozyme E ssentially all cellular processes that are mediated by structured RNAs also require one or more DExD͞H-box proteins (1). These proteins use the energy from ATP binding and hydrolysis to accelerate RNA structural transitions, which can represent folding steps toward the native state or conformational switches between functional forms. The requirement for proteins presumably arises because RNA base pairs and other local structure can be highly stable even in the absence of enforcing structure, such that folding steps or rearrangements that require significant unfolding require assistance to proceed efficiently (2-4).Despite their ubiquitous presence, key questions about the functions of DExD͞H-box proteins remain largely unanswered. First, what interactions direct different DExD͞H-box proteins to their physiological substrates? All of these proteins share a core ''helicase'' domain containing a set of conserved motifs, and most have additional domains, a few of which have been shown to recognize substrate RNAs or RNA-protein complexes (reviewed in ref. 5). On this basis, targeting has been proposed as a general role for these domains, and the specific interactions that target one DExD͞H-box protein have been delineated (6-9). Nevertheless, in general, the interactions that direct DExD͞H box proteins to their substrates remain to be identified.Second, h...

“…However, the list of proteins involved in early steps of SSU biogenesis is not complete, as new components of early pre-rRNA processing complexes continue to be identified (4,(14)(15)(16)(17). Here we add two previously undescribed proteins to that list, Utp23 and Utp24, based on the following lines of evidence: First, conditional depletion of either Utp23 or Utp24 specifically inhibits the A 0 -A 2 cleavages essential for SSU biogenesis ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The A 0 , A 1 , and A 2 pre-rRNA cleavages require a large ribonucleoprotein complex, the SSU processome͞90S preribosome, which contains at least 40 different proteins and numerous small nucleolar RNAs (snoRNAs) (1)(2)(3)(4). This complex likely corresponds to the terminal knobs seen in Miller chromatin spreads (1,5).…”

mentioning

confidence: 99%

The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation

Bleichert

Granneman

Osheim

et al. 2006

104

Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A 1 and͞or A2.ribosome biogenesis ͉ small subunit processome ͉ endonuclease ͉ yeast ͉ RNA processing R ibosomes translate mRNA into proteins and, thereby, govern cell growth and survival. In Saccharomyces cerevisiae, ribosome biogenesis starts with the transcription of the rDNA into a polycistronic rRNA precursor by RNA polymerase I in the nucleolus. The primary transcript, the 35S pre-rRNA, encodes the small subunit (SSU) rRNA, 18S, the large subunit rRNAs, 25S and 5.8S, and also external and internal transcribed spacer regions (5ЈETS, ITS1, ITS2, and 3ЈETS; Fig. 1). This precursor is extensively endoand exonucleolytically cleaved to mature the rRNAs. Maturation begins with endonucleolytic cleavages at sites A 0 , A 1 , and A 2 , the latter separating the 20S pre-rRNA, the direct precursor to the mature 18S rRNA, and the 27SA 2 pre-rRNA, the precursor to the large subunit rRNAs. The 20S pre-rRNA is exported into the cytoplasm as part of the 43S preribosome, where the 18S rRNA is matured by cleavage at site D.The A 0 , A 1 , and A 2 pre-rRNA cleavages require a large ribonucleoprotein complex, the SSU processome͞90S preribosome, which contains at least 40 different proteins and numerous small nucleolar RNAs (snoRNAs) (1-4). This complex likely corresponds to the terminal knobs seen in Miller chromatin spreads (1, 5). Depletion of essential SSU processome components prevents cleavages at sites A 0 -A 2 but not A 3 . Although this results in accumulation of the 23S pre-rRNA and inhibition of 18S synthesis, maturation of the 25S and 5.8S rRNAs can occur normally (Fig. 1).Despite the identification of numerous proteins involved in early pre-18S rRNA processing by large-scale purifications, the endonuclease(s) and possible candidates that carry out these cleavages have remained elusive. Recently, an interesting group of proteins possessing a PIN domain (PilT N terminus, PINc domain in SMART database) has been impl...