Non-conventional mRNA Splicing Research Articles

Detecting the Non-conventional mRNA Splicing and Translational Activation of HAC1 in Budding Yeast.

Protein-folding homeostasis in the endoplasmic reticulum (ER) is maintained by the unfolded protein response (UPR). UPR in Saccharomyces cerevisiae is regulated by a bZIP transcription factor, Hac1p. Under non-stress condition, HAC1 mRNA is translationally repressed. When un- or mis-folded proteins accumulate in the ER, HAC1 mRNA undergoes non-conventional mRNA splicing. The spliced HAC1 mRNA is translationally active and produces functional Hac1p, which initiates a transcriptional response that restores ER protein-folding homeostasis. Thus, the activation of yeast UPR is tightly regulated by HAC1 mRNA splicing. Here, we describe two methods that are used to monitor the splicing and translational status of HAC1 mRNA in budding yeast.

Protomer alignment modulates specificity of RNA substrate recognition by Ire1.

The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs-non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. Previously, we showed that the functional specialization lies in Ire1's RNase activity, which is either stringently splice-site specific or promiscuous (Li et al., 2018). Here, we developed an assay that reports on Ire1's RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain's dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.

Splicing HAC1/XBP1 mRNAs in Cytoplasm: The Non-Conventional mRNA Splicing Reaction in the Unfolded Protein Response

The majority of the secretory and transmembrane proteins are folded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, a collective of signalling pathways, termed the unfolded protein response (UPR), are activated to restore the ER protein folding homeostasis. The most evolutionarily conserved branch of UPR is mediated by the kinase/endoribonuclease Ire1. Ire1 mediates a cytosolic non-conventional mRNA splicing reaction of HAC1 mRNA in yeast and XBP1 mRNA in mammalian cells. The spliced HAC1/XBP1 mRNA is translated and produces a functional transcription factor, which initiates a transcriptional response to restore the protein folding homeostasis. The HAC1/XBP1 mRNA splicing reaction is biochemically distinct from the ones that are catalyzed by the spliceosome. Here, we review recent studies that provide a mechanistic understanding of the non-conventional mRNA splicing reaction.

TRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay.

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that catalyzes exon-exon ligation during tRNA biogenesis and the non-conventional splicing of HAC1 mRNA during the unfolded protein response (UPR). The UPR regulates the protein folding capacity of the endoplasmic reticulum (ER). ER stress activates Ire1, an ER-resident kinase/RNase, which excises an intron from HAC1 mRNA followed by exon-exon ligation by Trl1. The spliced product encodes for a potent transcription factor that drives the UPR. Here we report the crystal structure of Trl1 RNA ligase domain from Chaetomium thermophilum at 1.9 Å resolution. Structure-based mutational analyses uncovered kinetic competition between RNA ligation and degradation during HAC1 mRNA splicing. Incompletely processed HAC1 mRNA is degraded by Xrn1 and the Ski/exosome complex. We establish cleaved HAC1 mRNA as endogenous substrate for ribosome-associated quality control. We conclude that mRNA decay and surveillance mechanisms collaborate in achieving fidelity of non-conventional mRNA splicing during the UPR.

Engineering ER-stress dependent non-conventional mRNA splicing.

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un- or misfolded proteins. The ER membrane-resident kinase/RNase Ire1 maintains ER protein homeostasis through two fundamentally distinct processes. First, Ire1 can initiate a transcriptional response through a non-conventional mRNA splicing reaction to increase the ER folding capacity. Second, Ire1 can decrease the ER folding burden through selective mRNA decay. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two Ire1 functions have been evolutionarily separated. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we engineered an S. pombe Ire1 cleavage substrate into a splicing substrate, which confers S. pombe with both Ire1 functional outputs.

Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.

Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.

RTCB-1 mediates neuroprotection via XBP-1 mRNA splicing in the unfolded protein response pathway.

Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, is characterized by the degeneration of dopamine (DA) neurons and age-dependent formation of protein inclusions that contain the α-synuclein (α-syn) protein. RNA interference (RNAi) screening using Caenorhabditis elegans identified RTCB-1, an uncharacterized gene product, as one of several significant modifiers of α-syn protein misfolding. RTCB-1 is the worm ortholog of the human HSPC117 protein, a component of RNA trafficking granules in mammalian neurons. Here we show that RTCB-1 protects C. elegans DA neurons from age-dependent degeneration induced by human α-syn. Moreover, neuronal-specific RNAi depletion of rtcb-1 enhanced α-syn-induced degeneration. Similar results were obtained when worms were exposed to the DA neurotoxin 6-hydroxydopamine. HSPC117 has been characterized recently as an essential subunit of the human tRNA splicing ligase complex. tRNA ligases have alternative functions in RNA repair and nonconventional mRNA splicing events. For example, in yeast, unconventional splicing of HAC1, a transcription factor that controls the unfolded protein response (UPR), is mediated by a tRNA ligase. In C. elegans, we demonstrate that RTCB-1 is necessary for xbp-1 (worm homolog of HAC1) mRNA splicing. Moreover, using a RNA ligase-dead mutant, we determine that the ligase activity of worm RTCB-1 is required for its neuroprotective role, which, in turn, is mediated through XBP-1 in the UPR pathway. Collectively, these studies highlight the mechanistic intersection of RNA processing and proteostasis in mediating neuroprotection.

Diversity and roles of (t)RNA ligases

The discovery of discontiguous tRNA genes triggered studies dissecting the process of tRNA splicing. As a result, we have gained detailed mechanistic knowledge on enzymatic removal of tRNA introns catalyzed by endonuclease and ligase proteins. In addition to the elucidation of tRNA processing, these studies facilitated the discovery of additional functions of RNA ligases such as RNA repair and non-conventional mRNA splicing events. Recently, the identification of a new type of RNA ligases in bacteria, archaea, and humans closed a long-standing gap in the field of tRNA processing. This review summarizes past and recent findings in the field of tRNA splicing with a focus on RNA ligation as it preferentially occurs in archaea and humans. In addition to providing an integrated view of the types and phyletic distribution of RNA ligase proteins known to date, this survey also aims at highlighting known and potential accessory biological functions of RNA ligases.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-0944-2) contains supplementary material, which is available to authorized users.

Structural and functional basis for RNA cleavage by Ire1

BackgroundThe unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis.ResultsThis analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase.ConclusionsOur data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity

Accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). Ire1, an ER-resident transmembrane kinase/RNase, senses the protein folding status inside the ER. When activated, Ire1 oligomerizes and trans-autophosphorylates, activating its RNase and initiating a nonconventional mRNA splicing reaction. Splicing results in production of the transcription factor Hac1 that induces UPR target genes; expression of these genes restores ER homeostasis by increasing its protein folding capacity and allows abatement of UPR signaling. Here, we uncouple Ire1's RNase from its kinase activity and find that cells expressing kinase-inactive Ire1 can regulate Ire1's RNase, splice HAC1 mRNA, produce Hac1 protein, and induce UPR target genes. Unlike wild-type IRE1, kinase-inactive Ire1 cells display defects in Ire1 deactivation. Failure to properly inactivate Ire1 causes chronic ER stress and reduces cell survival under UPR-inducing conditions. Thus, Ire1-catalyzed phosphoryl-transfer aids disassembly of Ire1 signaling complexes and is a critical component of the UPR homeostatic feedback loop.

The unfolded protein response signals through high-order assembly of Ire1

Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-A crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors.

Genome-scale approaches for discovering novel nonconventional splicing substrates of the Ire1 nuclease

Three different genome-scale screens indicate that the HAC1 mRNA is the only substrate for the Ire1 nuclease in yeast.