Cytosolic Ribosomal Protein Haploinsufficiency affects Mitochondrial Morphology and Respiration.

The interplay between ribosomal protein composition and mitochondrial function is essential for sustaining energy homeostasis. Precise stoichiometric production of ribosomal proteins is crucial to maximize protein synthesis efficiency while reducing the energy costs to the cell. However, the impact of this balance on mitochondrial ATP generation, morphology and function remains unclear. Particularly, the loss of a single copy ribosomal protein gene is observed in Mendelian disorders like Diamond Blackfan Anemia and is common in somatic tumors, yet the implications of this imbalance on mitochondrial function and energy dynamics are still unclear. In this study, we investigated the impact of haploinsufficiency for four ribosomal protein genes implicated in ribosomopathy disorders (rps-10, rpl-5, rpl-33, rps-23) in Caenorhabditis elegans and corresponding reductions in human lymphoblast cells. Our findings uncover significant, albeit variably penetrant, mitochondrial morphological differences across these mutants, alongside an upregulation of glutathione transferases, and SKN-1 dependent increase in oxidative stress resistance, indicative of increased ROS production. Specifically, loss of a single copy of rps-10 in C. elegans led to decreased mitochondrial activity, characterized by lower energy levels and reduced oxygen consumption. A similar reduction in mitochondrial activity and energy levels was observed in human leukemia cells with a 50% reduction in RPS10 transcript levels. Importantly, we also observed alterations in the translation efficiency of nuclear and mitochondrial electron transport chain components in response to reductions in ribosomal protein genes' expression in both C. elegans and human cells. This suggests a conserved mechanism whereby the synthesis of components vital for mitochondrial function are adjusted in the face of compromised ribosomal machinery. Finally, mitochondrial membrane and cytosolic ribosomal components exhibited significant covariation at the RNA and translation efficiency level in lymphoblastoid cells across a diverse group of individuals, emphasizing the interplay between the protein synthesis machinery and mitochondrial energy production. By uncovering the impact of ribosomal protein haploinsufficiency on the translation efficiency of electron transport chain components, mitochondrial physiology, and the adaptive stress responses, we provide evidence for an evolutionarily conserved strategy to safeguard cellular functionality under genetic stress.

Integrated multiplexed assays of variant effect reveal determinants of catechol-O-methyltransferase gene expression.

Multiplexed assays of variant effect are powerful methods to profile the consequences of rare variants on gene expression and organismal fitness. Yet, few studies have integrated several multiplexed assays to map variant effects on gene expression in coding sequences. Here, we pioneered a multiplexed assay based on polysome profiling to measure variant effects on translation at scale, uncovering single-nucleotide variants that increase or decrease ribosome load. By combining high-throughput ribosome load data with multiplexed mRNA and protein abundance readouts, we mapped the cis-regulatory landscape of thousands of catechol-O-methyltransferase (COMT) variants from RNA to protein and found numerous coding variants that alter COMT expression. Finally, we trained machine learning models to map signatures of variant effects on COMT gene expression and uncovered both directional and divergent impacts across expression layers. Our analyses reveal expression phenotypes for thousands of variants in COMT and highlight variant effects on both single and multiple layers of expression. Our findings prompt future studies that integrate several multiplexed assays for the readout of gene expression.

Cellular targets and lysine selectivity of the HERC5 ISG15 ligase.

ISG15 is a type I interferon-induced ubiquitin-like modifier that functions in innate immune responses. The major human ISG15 ligase is hHERC5, a ribosome-associated HECT E3 that broadly ISGylates proteins cotranslationally. Here, we characterized the hHERC5-dependent ISGylome and identified over 2,000 modified lysines in over 1,100 proteins in IFN-ß-stimulated cells. In parallel, we compared the substrate selectivity hHERC5 to the major mouse ISG15 ligase, mHERC6, and analysis of sequences surrounding ISGylation sites revealed that hHERC5 and mHERC6 have distinct preferences for amino acid sequence context. Several features of the datasets were consistent with ISGylation of ribosome-tethered nascent chains, and mHERC6, like hHERC5, cotranslationally modified nascent polypeptides. The ISGylome datasets presented here represent the largest numbers of protein targets and modification sites attributable to a single Ub/Ubl ligase and the lysine selectivities of the hHERC5 and mHERC6 enzymes may have implications for the activities of HECT domain ligases, generally.

An inappropriate decline in ribosome levels drives a diverse set of neurodevelopmental disorders.

Many neurodevelopmental defects are linked to perturbations in genes involved in housekeeping functions, such as those encoding ribosome biogenesis factors. However, how reductions in ribosome biogenesis can result in tissue and developmental specific defects remains a mystery. Here we describe new allelic variants in the ribosome biogenesis factor AIRIM primarily associated with neurodevelopmental disorders. Using human cerebral organoids in combination with proteomic analysis, single-cell transcriptome analysis across multiple developmental stages, and single organoid translatome analysis, we identify a previously unappreciated mechanism linking changes in ribosome levels and the timing of cell fate specification during early brain development. We find ribosome levels decrease during neuroepithelial differentiation, making differentiating cells particularly vulnerable to perturbations in ribosome biogenesis during this time. Reduced ribosome availability more profoundly impacts the translation of specific transcripts, disrupting both survival and cell fate commitment of transitioning neuroepithelia. Enhancing mTOR activity by both genetic and pharmacologic approaches ameliorates the growth and developmental defects associated with intellectual disability linked variants, identifying potential treatment options for specific brain ribosomopathies. This work reveals the cellular and molecular origins of protein synthesis defect-related disorders of human brain development. Highlights: AIRIM variants reduce ribosome levels specifically in neural progenitor cells. Inappropriately low ribosome levels cause a transient delay in radial glia fate commitment.Reduced ribosome levels impair translation of a selected subset of mRNAs.Genetic and pharmacologic activation of mTORC1 suppresses AIRIM-linked phenotypes.

RiboGraph: An interactive visualization system for ribosome profiling data at read length resolution.

Ribosome profiling is a widely-used technique for measuring ribosome occupancy at nucleotide resolution. However, the need to analyze this data at nucleotide resolution introduces unique challenges in data visualization and analyses. In this study, we introduce RiboGraph, a dedicated visualization tool designed to work with .ribo files, a specialized and efficient format for ribosome occupancy data. Unlike existing solutions that rely on large alignment files and time-consuming preprocessing steps, RiboGraph operates on a purpose designed compact file type and eliminates the need for data preprocessing. This efficiency allows for interactive, real-time visualization at ribosome-protected fragment length resolution. By providing an integrated toolset, RiboGraph empowers researchers to conduct comprehensive visual analysis of ribosome occupancy data. Availability and Implementation: Source code, step-by-step installation instructions and links to documentation are available on GitHub: https://github.com/ribosomeprofiling/ribograph. On the same page, we provide test files and a step-by-step tutorial highlighting the key features of RiboGraph.

The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation.

Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel (PET) of the pre-60S subunit. Unlike canonical assembly factors, which associate exclusively with pre-60S subunits, we observed that Reh1 sediments with polysomes in addition to free 60S subunits. We therefore investigated the intriguing possibility that Reh1 remains associated with 60S subunits after the release of the anti-association factor Tif6 and after subunit joining. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes. Using selective ribosome profiling, we found that Reh1-bound ribosomes populate open reading frames near start codons. Reh1-bound ribosomes are also strongly enriched for initiator tRNA, indicating they are associated with early elongation events. Using single particle cryo-electron microscopy to image cycloheximide-arrested Reh1-bound 80S ribosomes, we found that Reh1-bound 80S contain A site peptidyl tRNA, P site tRNA and eIF5A indicating that Reh1 does not dissociate from 60S until early stages of translation elongation. We propose that Reh1 is displaced by the elongating peptide chain. These results identify Reh1 as the last assembly factor released from the nascent 60S subunit during its pioneer round of translation.

Integrated multiplexed assays of variant effect reveal cis-regulatory determinants of catechol-O-methyltransferase gene expression.

Multiplexed assays of variant effect are powerful methods to profile the consequences of rare variants on gene expression and organismal fitness. Yet, few studies have integrated several multiplexed assays to map variant effects on gene expression in coding sequences. Here, we pioneered a multiplexed assay based on polysome profiling to measure variant effects on translation at scale, uncovering single-nucleotide variants that increase and decrease ribosome load. By combining high-throughput ribosome load data with multiplexed mRNA and protein abundance readouts, we mapped the cis-regulatory landscape of thousands of catechol-O-methyltransferase (COMT) variants from RNA to protein and found numerous coding variants that alter COMT expression. Finally, we trained machine learning models to map signatures of variant effects on COMT gene expression and uncovered both directional and divergent impacts across expression layers. Our analyses reveal expression phenotypes for thousands of variants in COMT and highlight variant effects on both single and multiple layers of expression. Our findings prompt future studies that integrate several multiplexed assays for the readout of gene expression.

Single-cell quantification of ribosome occupancy in early mouse development.

Translation regulation is critical for early mammalian embryonic development1. However, previous studies had been restricted to bulk measurements2, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells.

Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADP ribosylation dependent manner to induce translational stalling.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.

satmut_utils: a simulation and variant calling package for multiplexed assays of variant effect.

The impact of millions of individual genetic variants on molecular phenotypes in coding sequences remains unknown. Multiplexed assays of variant effect (MAVEs) are scalable methods to annotate relevant variants, but existing software lacks standardization, requires cumbersome configuration, and does not scale to large targets. We present satmut_utils as a flexible solution for simulation and variant quantification. We then benchmark MAVE software using simulated and real MAVE data. We finally determine mRNA abundance for thousands of cystathionine beta-synthase variants using two experimental methods. The satmut_utils package enables high-performance analysis of MAVEs and reveals the capability of variants to alter mRNA abundance.

Developing high-affinity decoy receptors to treat multiple myeloma and diffuse large B cell lymphoma.

Disease relapse and treatment-induced immunotoxicity pose significant clinical challenges for patients with hematological cancers. Here, we reveal distinctive requirements for neutralizing TNF receptor ligands APRIL and BAFF and their receptor activity in MM and DLBCL, impacting protein translation and production in MM cells and modulating the translation efficiency of the ATM interactor (ATMIN/ACSIZ). Therapeutically, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as an inhibitor of APRIL and BAFF. While wild-type sBCMA-Fc effectively blocked APRIL signaling in MM, it lacked activity in DLBCL due to its weak BAFF binding. To expand the therapeutic utility of sBCMA-Fc, we engineered an affinity-enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) 4- and 500-fold stronger in binding to APRIL and BAFF, respectively. The mutant sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, we also demonstrated an adequate toxicity profile and on-target mechanism of action in nonhuman primate studies.

Desmosterol and 7-dehydrocholesterol concentrations in post mortem brains of depressed people: The role of trazodone.

Major depressive disorder (MDD) is a common, disabling, and heterogeneous condition that responds unpredictably to current treatments. We previously showed an association between depressive symptoms and plasma concentrations of two cholesterol precursors, desmosterol and 7-dehydrocholesterol (7DHC). Here, we measured total cholesterol and sterol concentrations with mass spectrometry in postmortem brain samples from depressed and control subjects. Mean (±SEM) desmosterol concentration was 8.9 ± 0.97 ng/mg in the depressed versus 10.7 ± 0.72 ng/mg in the control group. The mean of the posterior probability distribution for the difference in desmosterol concentration between the two groups was 2.36 (95% highest density interval [HDI] 0.59-4.17). Mean 7DHC concentrations, 12.5 ± 4.1 ng/mg in the depressed versus 5.4 ± 0.74 ng/mg in the control group, were unlikely to be different (95% HDI, [-1.37-0.34]). We found that presence of trazodone in the peri-mortem toxicology screen accounted for the observed difference in desmosterol concentrations. We also observed extremely high 7DHC levels in all 4 subjects who had taken trazodone. Trazodone has been recently found to inhibit 7-dehydrocholesterol reductase and alter sterol concentrations in rodents, cell culture, human fibroblasts, and blood. In this study, we demonstrate for the first time that trazodone alters human brain sterol composition. Given congenital deficiency of 7-dehydrocholesterol reductase results in Smith-Lemli-Opitz syndrome, our findings support the hypothesis that this commonly used medication may have previously unappreciated risks.

Genes with 5' terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein.

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5' UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.

Loss of coordinated expression between ribosomal and mitochondrial genes revealed by comprehensive characterization of a large family with a rare Mendelian disorder.

Non-canonical intronic variants are a poorly characterized yet highly prevalent class of alterations associated with Mendelian disorders. Here, we report the first RNA expression and splicing analysis from a family whose members carry a non-canonical splice variant in an intron of RPL11 (c.396 +3A>G). This mutation is causative for Diamond Blackfan Anemia (DBA) in this family despite incomplete penetrance and variable expressivity. Our analyses revealed a complex pattern of disruptions with many novel junctions of RPL11. These include an RPL11 transcript that is translated with a late stop codon in the 3' untranslated region (3'UTR) of the main isoform. We observed that RPL11 transcript abundance is comparable among carriers regardless of symptom severity. Interestingly, both the small and large ribosomal subunit transcripts were significantly overexpressed in individuals with a history of anemia in addition to congenital abnormalities. Finally, we discovered that coordinated expression between mitochondrial components and RPL11 was lost in all carriers, which may lead to variable expressivity. Overall, this study highlights the importance of RNA splicing and expression analyses in families for molecular characterization of Mendelian diseases.

Genes with 5' terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein.

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-Seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation level. We discover a functionally-coherent subset of human genes are preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5' UTRs from TOP transcripts can drive preferential expression in the presence of NSP1. Finally, we found that LARP1, a key effector protein in the mTOR pathway may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.

RiboFlow, RiboR and RiboPy: an ecosystem for analyzing ribosome profiling data at read length resolution.

SUMMARY: Ribosome occupancy measurements enable protein abundance estimation and infer mechanisms of translation. Recent studies have revealed that sequence read lengths in ribosome profiling data are highly variable and carry critical information. Consequently, data analyses require the computation and storage of multiple metrics for a wide range of ribosome footprint lengths. We developed a software ecosystem including a new efficient binary file format named 'ribo'. Ribo files store all essential data grouped by ribosome footprint lengths. Users can assemble ribo files using our RiboFlow pipeline that processes raw ribosomal profiling sequencing data. RiboFlow is highly portable and customizable across a large number of computational environments with built-in capabilities for parallelization. We also developed interfaces for writing and reading ribo files in the R (RiboR) and Python (RiboPy) environments. Using RiboR and RiboPy, users can efficiently access ribosome profiling quality control metrics, generate essential plots and carry out analyses. Altogether, these components create a software ecosystem for researchers to study translation through ribosome profiling. AVAILABILITY AND IMPLEMENTATION: For a quickstart, please see https://ribosomeprofiling.github.io. Source code, installation instructions and links to documentation are available on GitHub: https://github.com/ribosomeprofiling. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Simultaneous RNA purification and size selection using on-chip isotachophoresis with an ionic spacer.

We present an on-chip method for the extraction of RNA within a specific size range from low-abundance samples. We use isotachophoresis (ITP) with an ionic spacer and a sieving matrix to enable size-selection with a high yield of RNA in the target size range. The spacer zone separates two concentrated ITP peaks, the first containing unwanted single nucleotides and the second focusing RNA of the target size range (2-35 nt). Our ITP method excludes >90% of single nucleotides and >65% of longer RNAs (>35 nt). Compared to size selection using gel electrophoresis, ITP-based size-selection yields a 2.2-fold increase in the amount of extracted RNAs within the target size range. We also demonstrate compatibility of the ITP-based size-selection with downstream next generation sequencing. On-chip ITP-prepared samples reveal higher reproducibility of transcript-specific measurements compared to samples size-selected by gel electrophoresis. Our method offers an attractive alternative to conventional sample preparation for sequencing with shorter assay time, higher extraction efficiency and reproducibility. Potential applications of ITP-based size-selection include sequencing-based analyses of small RNAs from low-abundance samples such as rare cell types, samples from fluorescence activated cell sorting (FACS), or limited clinical samples.

Maternal Ribosomes Are Sufficient for Tissue Diversification during Embryonic Development in C. elegans.

Caenorhabditis elegans provides an amenable system to explore whether newly composed ribosomes are required to progress through development. Despite the complex pattern of tissues that are formed during embryonic development, we found that null homozygotes lacking any of the five different ribosomal proteins (RPs) can produce fully functional first-stage larvae, with similar developmental competence seen upon complete deletion of the multi-copy ribosomal RNA locus. These animals, relying on maternal but not zygotic contribution of ribosomal components, are capable of completing embryogenesis. In the absence of new ribosomal components, the resulting animals are arrested before progression from the first larval stage and fail in two assays for postembryonic plasticity of neuronal structure. Mosaic analyses of larvae that are a mixture of ribosome-competent and non-competent cells suggest a global regulatory mechanism in which ribosomal insufficiency in a subset of cells triggers organism-wide growth arrest.

Plasma sterols and depressive symptom severity in a population-based cohort.

Convergent evidence strongly suggests major depressive disorder is heterogeneous in its etiology and clinical characteristics. Depression biomarkers hold potential for identifying etiological subtypes, improving diagnostic accuracy, predicting treatment response, and personalization of treatment. Human plasma contains numerous sterols that have not been systematically studied. Changes in cholesterol concentrations have been implicated in suicide and depression, suggesting plasma sterols may be depression biomarkers. Here, we investigated associations between plasma levels of 34 sterols (measured by mass spectrometry) and scores on the Quick Inventory of Depressive Symptomatology-Self Report (QIDS-SR16) scale in 3117 adult participants in the Dallas Heart Study, an ethnically diverse, population-based cohort. We built a random forest model using feature selection from a pool of 43 variables including demographics, general health indicators, and sterol concentrations. This model comprised 19 variables, 13 of which were sterol concentrations, and explained 15.5% of the variation in depressive symptoms. Desmosterol concentrations below the fifth percentile (1.9 ng/mL, OR 1.9, 95% CI 1.2-2.9) were significantly associated with depressive symptoms of at least moderate severity (QIDS-SR16 score ≥10.5). This is the first study reporting a novel association between plasma concentrations cholesterol precursors and depressive symptom severity.

A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and N1-methyladenosine modification.

Introns are found in 5' untranslated regions (5'UTRs) for 35% of all human transcripts. These 5'UTR introns are not randomly distributed: Genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5'UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To model and understand the connection between coding-region sequence and 5'UTR intron status, we developed a classifier that can predict 5'UTR intron status with >80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5' proximal-intron-minus-like-coding regions ("5IM" transcripts). Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is enriched for non-AUG start codons, more extensive secondary structure both preceding the start codon and near the 5' cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the exon junction complex (EJC) at noncanonical 5' proximal positions. Finally, N1-methyladenosines are specifically enriched in the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence of a distinct 5IM class comprising ∼20% of human transcripts. This class is defined by depletion of 5' proximal introns, presence of specific RNA sequence features associated with low translation efficiency, N1-methyladenosines in the early coding region, and enrichment for noncanonical binding by the EJC.