John Paul Donohue

Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43

We used cross-linking and immunoprecipitation coupled with high-throughput sequencing to identify binding sites in 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein that, when mutated, causes amyotrophic lateral sclerosis. Massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs were changed (including Fus (Tls), progranulin and other transcripts encoding neurodegenerative disease–associated proteins) and 965 altered splicing events were detected (including in sortilin, the receptor for progranulin) following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels were most depleted by reduction in TDP-43 were derived from genes with very long introns and that encode proteins involved in synaptic activity. Lastly, we found that TDP-43 autoregulates its synthesis, in part by directly binding and enhancing splicing of an intron in the 3′ untranslated region of its own transcript, thereby triggering nonsense-mediated RNA degradation.

Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell–derived human neurons and in TDP-43 aggregate–containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.

Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUGexp RNA). To test whether CUGexp RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTGexp transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for ∼100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUGexp RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUGexp RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUGexp RNA causes two separate effects: loss of Mbnl1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.

Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation.

Introduction: One of the most critical and complex steps of protein synthesis is the coupled translocation of mRNA and tRNAs (mRNA and tRNAs) through the ribosome, catalyzed by the guanosine triphosphatase (GTPase) elongation factor EF-G. Although several of the main steps have been identified, the underlying molecular mechanisms of translocation are poorly understood. A central question is how structural rearrangements in the ribosome are coupled to movement of mRNA and tRNA. Methods: We trapped and crystallized complexes of Thermus thermophilus ribosomes bound with EF-G, mRNA, and tRNA, using the antibiotic fusidic acid (which prevents release of EF-G after GTP hydrolysis) or the nonhydrolyzable GTP analog GDPNP, in intermediate states of translocation. Their crystal structures were determined to resolutions from 3.5 to 4.1 Å. Results: The structures of the fusidic acid complex (Fus) and two GDPNP complexes (GDPNP-I and GDPNP-II) reveal conformational changes occurring during intermediate states of translocation, including large-scale (15° to 18°) rotation of the 30S subunit head and 3° to 5° rotation of the 30S body. In all complexes, the tRNA acceptor end has moved from the 50S subunit P site to the 50S E site, while the anticodon stem loop (ASL) and mRNA move with the head of the 30S subunit to positions between the P and E sites, forming chimeric pe*/E intermediate states. The elongated, mobile domain IV of EF-G moves to contact the head of the 30S subunit and the backbone of the mRNA. Two universally conserved bases of 16S rRNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. In the GDPNP complexes, structuring of the conserved switch loop I segment, which was disordered in previous structures, completes the cage that encloses GDPNP and fixes the relative geometry of EF-G domains I, III, and V. In the Fus complex, the position of fusidic acid overlaps that of switch loop I, stabilizing contacts between domains I and III that are normally made by the structured switch loop. Conclusion: Our structures capture intermediate states of the rate-limiting step of translocation, in which movement of the tRNA ASL and mRNA is coupled to rotational movement of the 30S subunit head. Slippage of the translational reading frame during reverse rotation of the head during translocation may be prevented by intercalation of bases C1397 and A1503 of 16S rRNA, which project from the body of the 30S subunit, between mRNA bases. The antibiotic fusidic acid appears to stabilize binding of EF-G to the ribosome in the GDP state by mimicking the structure of the conserved core of switch loop I of EF-G in the GTP state. Revealed in Translation The ribosome, with the help of transfer RNAs (tRNAs), converts the triple genetic code in messenger RNA (mRNA) into protein. Upon decoding of a codon, the mRNA and associated tRNAs must be moved through the ribosome, so that the next codon can be read, with a new charged tRNA taken in at the A (aminoacyl-tRNA) site, the newly extended peptidyl-tRNA moved into the P (peptidyl-tRNA) site, and the deacylated tRNA removed from the exit site in the ribosome (see the Perspective by Rodnina). Crystal structures from Tourigny et al. (p. 1235490), Pulk and Cate (p. 1235970), and Zhou et al. (p. 1236086), variously capture the prokaryotic ribosome during this translocation phase, revealing the hybrid states of the tRNAs and the substantial motions of the 30S ribosomal subunit during the process, the role of elongation factor G, and suggest how the direction and reading frame of the mRNA is maintained. Crystal structures reveal how messenger RNA and transfer RNAs transition through the prokaryotic ribosome during translation. [Also see Perspective by Rodnina] Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA–EF-G complexes trapped with β,γ-imidoguanosine 5′-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.

How the ribosome hands the A-site tRNA to the P site during EF-G–catalyzed translocation

Caught in the act of making protein The ribosome is a large RNA-protein complex that converts the genetic code stored in messenger RNA (mRNA) into proteins. Zhou et al. have determined the structure of a bacterial ribosome caught in the act of decoding an mRNA. Transfer RNAs (tRNAs) decipher the genetic code in the mRNA to ensure that the ribosome uses the correct amino acids. The structure shows tRNAs in the process of being moved between successive protein-building binding pockets as the ribosome reads the mRNA like a piece of old-fashion computer tape. Science, this issue p. 1188 The structure of an intermediate shows how the ribosome moves transfer RNAs from one binding site to another. Coupled translocation of messenger RNA and transfer RNA (tRNA) through the ribosome, a process catalyzed by elongation factor EF-G, is a crucial step in protein synthesis. The crystal structure of a bacterial translocation complex describes the binding states of two tRNAs trapped in mid-translocation. The deacylated P-site tRNA has moved into a partly translocated pe/E chimeric hybrid state. The anticodon stem-loop of the A-site tRNA is captured in transition toward the 30S P site, while its 3′ acceptor end contacts both the A and P loops of the 50S subunit, forming an ap/ap chimeric hybrid state. The structure shows how features of ribosomal RNA rearrange to hand off the A-site tRNA to the P site, revealing an active role for ribosomal RNA in the translocation process.

Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing

During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations, prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and that pre-messenger RNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s), but also on those of competing pre-mRNAs. Competition between RNAs for limiting processing factors appears to be a general condition in eukaryotes for a variety of posttranscriptional control mechanisms including microRNA (miRNA) repression, polyadenylation, and splicing.

Molecular mechanics of 30S subunit head rotation

Significance Ribosomes are the site of protein synthesis in all cells. Ribosomal polypeptide synthesis involves reading the information encoded in the mRNA and adding the correct amino acid via a tRNA. Elongation of the polypeptide chain proceeds through translocation, where the ribosome couples the movement of the mRNA and its associated tRNAs by precisely one codon. Translocation requires large-scale movement of the small-subunit head domain of the ribosome. By comparing 55 ribosome structures, we show that movement universally results from coupled hinging at two separate loci in the small subunit. This mechanism explains the mode of action of the antibiotic spectinomycin and suggests a means by which the ribosome controls translocation. During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2.

Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

Internal ribosome entry site (IRES) RNAs are elements of viral or cellular mRNAs that bypass steps of canonical eukaryotic cap-dependent translation initiation. Understanding of the structural basis of IRES mechanisms is limited, partially due to a lack of high-resolution structures of IRES RNAs bound to their cellular targets. Prompted by the universal phylogenetic conservation of the ribosomal P site, we solved the crystal structures of proposed P site binding domains from two intergenic region IRES RNAs bound to bacterial 70S ribosomes. The structures show that these IRES domains nearly perfectly mimic a tRNA•mRNA interaction. However, there are clear differences in the global shape and position of this IRES domain in the intersubunit space compared to those of tRNA, supporting a mechanism for IRES action that invokes hybrid state mimicry to drive a noncanonical mode of translocation. These structures suggest how relatively small structured RNAs can manipulate complex biological machines.

Protein-RNA Networks Regulated by Normal and ALS-Associated Mutant HNRNPA2B1 in the Nervous System

HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide crosslinking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within ∼2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1. VIDEO ABSTRACT.

RNA-binding protein CPEB1 remodels host and viral RNA landscapes

Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3′ untranslated regions (3′ UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.