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The metastatic lymphatic drainage of testis cancer to the retroperitoneum was noted clinically about a century ago. Beginning with extraperitoneal approaches, RPLND was attempted. The first cure after RPLND of node positive disease was in 1905 by Cuneo in Paris. Transperitoneal approaches failed due to infection until post World War II experience at Walter Reed Army Hospital. Thoracoabdominal approaches became popular several decades later. But Improved exposure and vascular management strategies led to increased usage of the transabdominal approach once again. The advent of platinum based combination chemotherapy has had a major impact on both the timing of and the technical requirements of RPLND. Owing to our early involvement in this area, we have accumulated the largest database available on this disease. Our experience with over 2500 RPLNDs in the last 3 decades is divided between low stage (I and II) and high stage (III, postchemotherapy) disease. The former has been "down-regulated" to modified templates and prospective nerve sparing techniques to preserve ejaculation. The latter has been "up-regulated" to include a spectrum of surgical needs including hepatic, vascular, gut and mediastinal resections. Despite these extended requirements, outcomes are good (> 80% survival) postchemotherapy. The evolutionary change of RPLND reflects an optimal paradigm of surgical-medical oncologic interaction. [ABSTRACT FROM AUTHOR]

Recognition of the intron branchpoint during spliceosome assembly is a multistep process that defines both mRNA structure and amount. A branchpoint sequence motif UACUAAC is variably conserved in eukaryotic genomes, but in some organisms more than one protein can recognize it. Here we show that SF1 and Quaking (QKI) compete for a subset of intron branchpoints with the sequence ACUAA. SF1 activates exon inclusion through this sequence, but QKI represses the inclusion of alternatively spliced exons with this intron branchpoint sequence. Using mutant reporters derived from a natural intron with two branchpoint-like sequences, we find that when either branchpoint sequence is mutated, the other is used as a branchpoint, but when both are present, neither is used due to high affinity binding and strong splicing repression by QKI. QKI occupancy at the dual branchpoint site directly prevents SF1 binding and subsequent recruitment of spliceosome-associated factors. Finally, the ectopic expression of QKI in budding yeast (which lacks QKI ) is lethal, due at least in part to widespread splicing repression. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of branchpoint sequences that closely mirror its high affinity binding site. This suggests that QKI and degenerate branchpoint sequences may have co-evolved as a means through which specific gene expression patterns could be maintained in QKI-expressing or non-expressing cells in metazoans, plants, and animals.

Loss of the translational reading frame leads to misincorporation and premature termination, which can have lethal consequences. Based on structural evidence that A1503 of 16S rRNA intercalates between specific mRNA bases, we tested the possibility that it plays a role in maintenance of the reading frame by constructing ribosomes with an abasic nucleotide at position 1503. This was done by specific cleavage of 16S rRNA at position 1493 using the colicin E3 endonuclease and replacing the resulting 3'-terminal 49mer fragment with a synthetic oligonucleotide containing the abasic site using a novel splinted RNA ligation method. Ribosomes reconstituted from the abasic 1503 16S rRNA were highly active in protein synthesis but showed elevated levels of spontaneous frameshifting into the -1 reading frame. We then asked whether the residual frameshifting persisting in control ribosomes containing an intact A1503 is due to the absence of the N6-dimethyladenosine modifications at positions 1518 and 1519. Indeed, this frameshifting was rescued by site-specific methylation in vitro by the ksgA methylase. These findings thus implicate two different sites near the 3' end of 16S rRNA in maintenance of the translational reading frame, providing yet another example of a functional role for ribosomal RNA in protein synthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Rare, full-length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envisioned and tested a hypothesis for their formation using Saccharomyces cerevisiae , documenting full-length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron lariat spliceosome (ILS) in which the 3'-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3' processing machinery) attacks the branch, joining the intron 3' end to the 5' splice site in a 3'-5' linked circle. Human U2 and U12 spliceosomes produce analogous full-length and processed circles. Postsplicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution., (© 2024 Ares et al.; Published by Cold Spring Harbor Laboratory Press.)

Intron branchpoint (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect the binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after the addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during cotranscriptional splicing in Plad-B using single-molecule intron tracking to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between the binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten the characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy., (© 2024 Hunter et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3'-terminal pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ribosomal RNAs, along with their substrates the transfer RNAs, contain the most highly conserved nucleotides in all of biology. We have assembled a database containing structure-based alignments of sequences of the small-subunit rRNAs from organisms that span the entire phylogenetic spectrum, to identify the nucleotides that are universally conserved. In its simplest (bacterial and archaeal) forms, the small-subunit rRNA has ∼1500 nt, of which we identify 140 that are absolutely invariant among the 1961 species in our alignment. We examine the positions and detailed structural and functional interactions of these universal nucleotides in the context of a half century of biochemical and genetic studies and high-resolution structures of ribosome functional complexes. The vast majority of these nucleotides are exposed on the subunit interface surface of the small subunit, where the functional processes of the ribosome take place. However, only 40 of them have been directly implicated in specific ribosomal functions, such as contacting the tRNAs, mRNA, or translation factors. The roles of many other invariant nucleotides may serve to constrain the positions and orientations of those nucleotides that are directly involved in function. Yet others can be rationalized by participation in unusual noncanonical tertiary structures that may uniquely allow correct folding of the rRNA to form a functional ribosome. However, there remain at least 50 nt whose universal conservation is not obvious, serving as a metric for the incompleteness of our understanding of ribosome structure and function., (© 2022 Noller et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Objectives/hypothesis: Bilateral myringotomy and tympanostomy tube placement (BMT) is the most common pediatric surgery in the United States. Intraoperative middle ear effusion (MEE) is a risk factor for future BMTs in children with recurrent acute otitis media (RAOM). However, the impact of the type of MEE is unknown. Here, we assess otologic outcomes based on intraoperative MEE type and indication for surgery., Study Design: Case series chart review., Methods: After institutional review board approval, we performed a review of children undergoing BMTs between 2008 and 2009. Included patients had their first BMT, preoperative visit, and an operative report. Patients with cleft palate or Down syndrome were excluded. Indications for surgery included RAOM and chronic otitis media with effusion (COME). Other variables evaluated were future BMT, acquired cholesteatoma, and otorrhea. Logistic regression was used for statistical analysis., Results: Out of 1,045 patients reviewed, 680 were included and underwent their first BMT. There were 619 patients who had RAOM. Serous effusions were present in 22.2%, mucoid in 31.3%, purulent in 12.9%, undocumented or bloody in 2.3% of patients, and 31.2% of patients had dry middle ears. Moreover, 22.7% of patients underwent future BMTs. In RAOM patients, serous effusions decreased odds of perforation (odds ratio [OR]: 0.195, 95% confidence interval [CI]: 0.0438-0.867, P = .032), and purulent effusions increased the odds of in-office otorrhea suctioning (OR: 2.13, 95% CI: 1.20-3.77, P = .010) compared to dry. Mucoid effusions had no significant effect on outcomes in COME or RAOM patients., Conclusions: Intraoperative MEEs were noted in 68.7% of cases; purulent effusions increase the odds of in-office suctioning in RAOM patients., Level of Evidence: 4 Laryngoscope, 131:E993-E997, 2021., (© 2020 The American Laryngological, Rhinological and Otological Society, Inc.)

[This corrects the article DOI: 10.1371/journal.pgen.1008249.].

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function., Competing Interests: The authors have declared that no competing interests exist.

The elongation factor G (EF-G)-catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Although the mRNA also undergoes movement, codon-anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (along with rotation of the 30S head domain) can occur in the absence of EF-G and GTP, EF-G is essential for enforcing coupled movement of the tRNAs and their mRNA codons to maintain the reading frame., Competing Interests: The authors declare no conflict of interest.

Quaking protein isoforms arise from a single Quaking gene and bind the same RNA motif to regulate splicing, translation, decay, and localization of a large set of RNAs. However, the mechanisms by which Quaking expression is controlled to ensure that appropriate amounts of each isoform are available for such disparate gene expression processes are unknown. Here we explore how levels of two isoforms, nuclear Quaking-5 (Qk5) and cytoplasmic Qk6, are regulated in mouse myoblasts. We found that Qk5 and Qk6 proteins have distinct functions in splicing and translation, respectively, enforced through differential subcellular localization. We show that Qk5 and Qk6 regulate distinct target mRNAs in the cell and act in distinct ways on their own and each other's transcripts to create a network of autoregulatory and cross-regulatory feedback controls. Morpholino-mediated inhibition of Qk translation confirms that Qk5 controls Qk RNA levels by promoting accumulation and alternative splicing of Qk RNA, whereas Qk6 promotes its own translation while repressing Qk5. This Qk isoform cross-regulatory network responds to additional cell type and developmental controls to generate a spectrum of Qk5/Qk6 ratios, where they likely contribute to the wide range of functions of Quaking in development and cancer., (© 2017 Fagg et al.; Published by Cold Spring Harbor Laboratory Press.)

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.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.

Glioblastoma multiforme (GBM) is an aggressive and nearly uniformly fatal malignancy of the central nervous system. Despite extensive research and clinical trials over the past 50 years, very little progress has been made to significantly alter its lethal prognosis. The current standard of care (SOC) includes maximal surgical resection, radiation therapy and chemotherapy and temozolomide (TMZ), including the selective use of glucocorticoids for symptom control. These same treatments, however, have the potential to create an environment that may actually facilitate tumor growth and survival. Research investigating the unique metabolic needs of tumor cells has led to the proposal of a new metabolic treatment for various cancers including GBMs that may enhance the effectiveness of the SOC. The goal of metabolic cancer therapy is to restrict GBM cells of glucose, their main energy substrate. By recognizing the underlying energy production requirements of cancer cells, newly proposed metabolic therapy is being used as an adjunct to standard GBM therapies. This review will discuss the calorie restricted ketogenic diet (CR-KD) as a promising potential adjunctive metabolic therapy for patients with GBMs. The effectiveness of the CR-KD is based on the "Warburg Effect" of cancer metabolism and the microenvironment of GBM tumors. We will review recent case reports, clinical studies, review articles, and animal model research using the CR-KD and explain the principles of the Warburg Effect as it relates to CR-KD and GBMs.

The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

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.

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., (Copyright © 2014, American Association for the Advancement of Science.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Bacterial translation termination is mediated by release factors RF1 and RF2, which recognize stop codons and catalyze hydrolysis of the peptidyl-tRNA ester bond. The catalytic mechanism has been debated. We proposed that the backbone amide NH group, rather than the side chain, of the glutamine of the universally conserved GGQ motif participates in catalysis by H-bonding to the tetrahedral transition-state intermediate and by product stabilization. This was supported by complete loss of RF1 catalytic activity when glutamine is replaced by proline, the only residue that lacks a backbone NH group. Here, we present the 3.4 Å crystal structure of the ribosome complex containing the RF2 Q253P mutant and find that its fold, including the GGP sequence, is virtually identical to that of wild-type RF2. This rules out proline-induced misfolding and further supports the proposal that catalytic activity requires interaction of the Gln-253 backbone amide with the 3' end of peptidyl-tRNA., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA ("STAR" motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.

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.

Bone was analyzed for ketamine and norketamine to examine whether different patterns of drug exposure could be discriminated. Rats received (intraperitoneally) one 75 mg/kg dose (Acute-1 and Acute-2 groups), three 25-mg/kg doses 1 hour apart (Repeated group), or nine single daily ketamine doses of 75 mg/kg followed by a 24-h washout period (Chronic group). Following euthanasia, all animals decomposed to skeleton outdoors. Ground samples of recovered bone underwent methanolic extraction and analysis by gas chromatography-mass spectrometry after solid-phase extraction. Drug levels (mass normalized response ratios) were compared across bone types and exposure pattern. Bone type significantly influenced drug level for the Acute-1 and Repeated dose groups, and the drug/metabolite level ratio (DMLR) for the Acute-1 group. Mean ketamine and norketamine level and DMLR varied by up to 8-fold, 7-fold and 3-fold, respectively, in the Acute-1 group, and by up to 24-fold, 5-fold and 10-fold, respectively, in the Repeated group. Drug level and DMLR differed significantly between the Acute-1 and Repeated groups for most bone types. In the Chronic group, only 1/16 and 4/16 samples were positive for ketamine and norketamine, respectively. All Acute-2 samples were positive for ketamine and norketamine. The Acute-2 and Chronic groups differed significantly in ketamine and norketamine levels, and DMLR.

Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

Skeletal tissues (rat) were analyzed for ketamine (KET) and norketamine (NKET) following acute ketamine exposure (75 mg/kg i.p.) to examine the influence of bone type and decomposition period on drug levels. Following euthanasia, drug-free (n = 6) and drug-positive (n = 20) animals decomposed outdoors in rural Ontario for 0, 1, or 2 weeks. Skeletal remains were recovered and ground samples of various bones underwent passive methanolic extraction and analysis by GC-MS after solid-phase extraction. Drug levels, expressed as mass normalized response ratios, were compared across tissue types and decomposition periods. Bone type was a main effect (p < 0.05) for drug level and drug/metabolite level ratio (DMLR) for all decomposition times, except for DMLR after 2 weeks of decomposition. Mean drug level (KET and NKET) and DMLR varied by up to 23-fold, 18-fold, and 5-fold, respectively, between tissue types. Decomposition time was significantly related to DMLR, KET level, and NKET level in 3/7, 4/7, and 1/7 tissue types, respectively. Although substantial sitedependence may exist in measured bone drug levels, ratios of drug and metabolite levels should be investigated for utility in discrimination of drug administration patterns in forensic work.

Introduction: AMG 108 is a fully human, immunoglobulin subclass G2 (IgG2) monoclonal antibody that binds the human interleukin-1 (IL-1) receptor type 1, inhibiting the activity of IL-1a and IL-1b. In preclinical studies, IL-1 inhibition was shown to be beneficial in models of osteoarthritis (OA). The purpose of this two-part study was to evaluate the safety and pharmacokinetics (PK; Part A) and clinical effect (Part B) of AMG 108 in a double-blind, placebo-controlled, multiple-dose study in patients with OA of the knee., Methods: In Part A, patients received placebo or AMG 108 subcutaneously (SC; 75 mg or 300 mg) or intravenously (IV; 100 mg or 300 mg) once every 4 weeks for 12 weeks; in Part B, patients received placebo or 300 mg AMG 108 SC, once every 4 weeks for 12 weeks. The clinical effect of AMG 108 was measured in Part B by using the Western Ontario and McMaster Universities (WOMAC) osteoarthritis index pain score., Results: In Part A, 68 patients were randomized, and 64 received investigational product. In Part B, 160 patients were randomized, and 159 received investigational product. AMG 108 was well tolerated. Most adverse events (AEs), infectious AEs, serious AEs and infections, as well as withdrawals from the study due to AEs occurred at similar rates in both active and placebo groups. One death was reported in an 80-year-old patient (Part A, 300 mg IV AMG 108; due to complications of lobar pneumonia). AMG 108 serum concentration-time profiles exhibited nonlinear PK. The AMG 108 group in Part B had statistically insignificant but numerically greater improvement in pain compared with the placebo group, as shown by the WOMAC pain scores (median change, -63.0 versus -37.0, respectively)., Conclusions: The safety profile of AMG 108 SC and IV was comparable with placebo in patients with OA of the knee. Patients who received AMG 108 showed statistically insignificant but numerically greater improvements in pain; however, minimal, if any, clinical benefit was observed., Trial Registration: This study is registered with ClinicalTrials.gov with the identifier NCT00110942.

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.

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.

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG(exp) RNA). To test whether CUG(exp) RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG(exp) transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for approximately 100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUG(exp) RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUG(exp) RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG(exp) 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.

Purpose: We determined outcomes in patients with testicular cancer with large volume (greater than 10 cm) retroperitoneal teratoma treated with post-chemotherapy retroperitoneal lymph node dissection., Materials and Methods: A retrospective review of our testicular cancer database was performed from 1995 to 2005 to identify patients undergoing post-chemotherapy retroperitoneal lymph node dissection for residual masses larger than 10 cm with final pathological examination revealing teratoma. A total of 99 patients met the study inclusion criteria., Results: A total of 27 patients presented with disease limited to the retroperitoneum, 46 had 2 or 3 disease sites and 26 had 4 or more disease sites. Mean and median hospital stay was 7.3 and 5.0 days, respectively. There were 23 recurrences in 27 locations with the most common being pulmonary in 5, mediastinal in 5 and retroperitoneal in 5. The 2 and 5-year disease-free survival was 86% and 75% with a mean followup of 42 months. The 2-year disease-free survival for patients presenting with retroperitoneal disease only was 86% compared to 79% and 41% for patients presenting with 2 to 3 disease sites and more than 4 disease sites, respectively (p = 0.004). The 2-year disease-free survival was 78% for patients undergoing retroperitoneal lymph node dissection alone, 80% for retroperitoneal lymph node dissection plus 1 or 2 other sites and 40% for retroperitoneal lymph node dissection plus resection of 3 or more disease sites (p = 0.026)., Conclusions: The recurrence rate for resected post-chemotherapy high volume teratoma is 25% at 5 years. The most common sites of recurrence are the lung, mediastinum and retroperitoneum.

As the genomes of more eukaryotic pathogens are sequenced, understanding how molecular differences between parasite and host might be exploited to provide new therapies has become a major focus. Central to cell function are RNA-containing complexes involved in gene expression, such as the ribosome, the spliceosome, snoRNAs, RNase P, and telomerase, among others. In this article we identify by comparative genomics and validate by RNA analysis numerous previously unknown structural RNAs encoded by the Plasmodium falciparum genome, including the telomerase RNA, U3, 31 snoRNAs, as well as previously predicted spliceosomal snRNAs, SRP RNA, MRP RNA, and RNAse P RNA. Furthermore, we identify six new RNA coding genes of unknown function. To investigate the relationships of the RNA coding genes to other genomic features in related parasites, we developed a genome browser for P. falciparum (http://areslab.ucsc.edu/cgi-bin/hgGateway). Additional experiments provide evidence supporting the prediction that snoRNAs guide methylation of a specific position on U4 snRNA, as well as predicting an snRNA promoter element particular to Plasmodium sp. These findings should allow detailed structural comparisons between the RNA components of the gene expression machinery of the parasite and its vertebrate hosts.

Background: Traditionally, postchemotherapy (PC) surgery for metastatic nonseminomatous germ cell tumor (NSGCT) has used a full bilateral retroperitoneal lymph node dissection (RPLND) from the crus of the diaphragm to the bifurcation of the common iliac arteries, from ureter to ureter. With the primary landing zone well defined in low-volume retroperitoneal disease, the authors performed modified dissections in the PC setting in a select population; and, herein, they report disease outcome., Methods: From 1991 to 2004, a retrospective review of the testicular cancer database at the authors' institution was performed to identify patients with NSGCT, normal serum tumor markers after cisplatin-based chemotherapy, and residual retroperitoneal tumor who underwent modified PC-RPLND. All patients had metastatic disease at initial presentation that was limited to the primary landing zone (left or right)., Results: One hundred patients were identified, including 43 who underwent a right modified template, 18 patients who underwent a left full modified template, and 39 patients who underwent a left modified template. Pathology revealed cancer in 2% of patients, teratoma in 62% of patients, and necrosis in 36% of patients. The 2- and 5-year disease-free survival rate was 95%, and the median follow-up was 31.9 months (range, 1-152 months). Four patients developed recurrent disease with a median time to recurrence of 8.25 months (range, 6-11 months). All recurrences were outside the boundaries of a full bilateral RPLND., Conclusions: Selected patients at PC surgery can be managed with modified PC-RPLND., ((c) 2007 American Cancer Society.)

Purpose: We defined the blood loss, operative time and short-term morbidity of primary retroperitoneal lymph node dissection in a contemporary series to assess whether laparoscopic retroperitoneal lymph node dissection actually confers the magnitude of benefit claimed., Materials and Methods: A retrospective chart review was performed of 75 consecutive patients who underwent primary retroperitoneal lymph node dissection during the 18 months ending May 2005. Two patients were excluded, including 1 who underwent right hemicolectomy for cecal adenocarcinoma and 1 with a pure seminomatous intra-abdominal testicle., Results: Of the 73 patients 69 (94%) underwent unilateral dissection and 60 (82.2%) underwent a nerve sparing procedure. Mean operative time was 132 minutes (range 81 to 246) and mean blood loss was 207 cc (range 50 to 500). Nasogastric tubes were placed in 2 patients (2.7%). Mean time to start clear liquids was 1.0 day. Mean hospital stay was 2.8 days (range 2 to 4)., Conclusions: The short-term morbidity of open retroperitoneal lymph node dissection, including operative time, blood loss and hospital stay, has significantly improved compared to historical controls. Perioperative management has changed with time. Comparing the morbidity of laparoscopic retroperitoneal lymph node dissection to that of historical controls is inappropriate.

Elevated serum tumor markers after cisplatin-based chemotherapy usually contraindicate surgery because of the presence of active germ-cell elements; however, some patients have undergone PCRPLND with curative intent. We evaluated the role of surgery to resect retroperitoneal-only marker positive tumor. Residual germ-cell cancer was identified in 50% of patients with elevated tumor markers with one third alive at 5 years; 5-year survival with residual teratoma or necrosis was 77.5% and 85.7%, respectively. Predictors of retroperitoneal teratoma or fibrosis included declining tumor makers at surgery, betaHCG < 100, and first-line chemotherapy. Predictors of death included rising preoperative betaHCG, elevated AFP, redo RPLND, and active germ-cell cancer in the resected specimen. Select patients with elevated tumor markers after chemotherapy are cured with surgery.

Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified>50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called "ultraconserved" elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell.

Objectives: To determine whether the size of the primary tumor and degree of preorchiectomy serum alpha-fetoprotein (AFP) and beta-human chorionic gonadotropin (beta-hCG) elevation predict for retroperitoneal pathologic findings in patients with clinical Stage A nonseminomatous germ cell tumor undergoing primary retroperitoneal lymph node dissection., Methods: The testicular cancer database was queried to identify patients with clinical Stage A nonseminomatous germ cell tumor with normalization of serum tumor markers after orchiectomy who had undergone retroperitoneal lymph node dissection. A total of 779 patients were identified. The preorchiectomy serum tumor marker level was recorded and categorized into the following subsets: AFP: less than 20 (normal), 20 to 100, 100 to 1000, and more than 1000 ng/dL; and beta-hCG: less than 5.0 (normal), 5 to 100, 100 to 1000, and more than 1000. The association between AFP, beta-hCG, and primary tumor size and retroperitoneal pathologic findings was determined., Results: The retroperitoneal pathologic examination revealed metastatic disease in 207 patients (26.6%). The preorchiectomy serum beta-hCG level, as a categorical variable, was not predictive of positive retroperitoneal pathologic findings (P = 0.187). The preorchiectomy serum AFP did predict for positive retroperitoneal pathologic findings, with lower serum AFP levels associated with a greater incidence of retroperitoneal metastasis (P <0.001). The primary tumor size was not predictive of positive retroperitoneal pathologic findings (P = 0.113)., Conclusions: Neither the primary tumor size nor the preorchiectomy beta-hCG level was predictive of retroperitoneal metastases. However, a normal preorchiectomy AFP level was associated with a greater incidence of retroperitoneal metastases.

Purpose: The presence of extranodal extension identified at primary retroperitoneal lymph node dissection has been associated with an increased risk of disease recurrence, and as such these patients are sometimes treated with adjuvant chemotherapy. We decided to evaluate the significance of extranodal extension on disease-free survival in patients with pathological stage B nonseminomatous germ cell tumor who did not receive adjuvant chemotherapy., Materials and Methods: A retrospective review of our testicular cancer database was performed to identify all patients with clinical stage A nonseminomatous germ cell tumor who underwent primary retroperitoneal lymph node dissection and were found to have retroperitoneal metastasis with 5 or fewer involved nodes and no metastatic node larger than 2 cm. No patient received adjuvant chemotherapy, and all had a minimum followup of 24 months. A single pathologist (LC), who was blinded to clinical outcome, reviewed the retroperitoneal nodal package to identify the presence or absence of extranodal extension, defined as cancer perforating through the lymph node capsule into perinodal tissue., Results: A total of 80 patients were identified with a median followup 48 months, and a 2 and 5-year disease-free survival of 75%. Extranodal extension was present in 23 patients and absent in 57 patients with a median followup of 54 and 44 months, respectively. The 5-year disease-free survival for patients with and without extranodal extension was 74% and 75%, respectively (p=0.67)., Conclusions: We were unable to detect any prognostic significance of extranodal extension in patients found to have retroperitoneal metastasis at primary retroperitoneal lymph node dissection.

With long-term survival in excess of 90% across all stages, testicular cancer has come to represent the model for successful multidisciplinary cancer care. Retroperitoneal lymph node dissection (RPLND) remains an integral component of testis cancer management strategies for both early- and advanced-stage disease. Commensurate with improvements made in clinical staging and in our understanding of the natural history of testis cancer, lymphatic spread, and neuroanatomy, considerable modifications in the technique and template of RPLND have taken place. The morbidity of primary RPLND and postchemotherapy RPLND is low when performed by experienced surgeons. This article reviews the evolution, role, and technique of RPLND in contemporary practice.

Increased serum tumor markers after cisplatin-based chemotherapy have usually been considered a contraindication to surgery because of the presence of persistent active germ cell elements. However, a select population of patients with elevated serum tumor markers have undergone post-chemotherapy retroperitoneal lymph node dissection (RPLND) with curative intent. We evaluated the role of surgery to resect retroperitoneal-only marker positive tumor. Long-term survival was observed in 50% of patients. Residual germ cell cancer was identified in 50% of patients, with a third alive at 5 years with no observed benefit from adjuvant chemotherapy. Select patients with increased tumor markers after chemotherapy are cured with surgery.