Tissue morphology influences the temporal program of human brain organoid development.

Progression through fate decisions determines cellular composition and tissue architecture, but how that same architecture may impact cell fate is less clear. We took advantage of organoids as a tractable model to interrogate this interaction of form and fate. Screening methodological variations revealed that common protocol adjustments impacted various aspects of morphology, from macrostructure to tissue architecture. We examined the impact of morphological perturbations on cell fate through integrated single nuclear RNA sequencing (snRNA-seq) and spatial transcriptomics. Regardless of the specific protocol, organoids with more complex morphology better mimicked in vivo human fetal brain development. Organoids with perturbed tissue architecture displayed aberrant temporal progression, with cells being intermingled in both space and time. Finally, encapsulation to impart a simplified morphology led to disrupted tissue cytoarchitecture and a similar abnormal maturational timing. These data demonstrate that cells of the developing brain require proper spatial coordinates to undergo correct temporal progression.

Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain.

Motile cilia defects impair cerebrospinal fluid (CSF) flow and can cause brain and spine disorders. The development of ciliated cells, their impact on CSF flow, and their function in brain and axial morphogenesis are not fully understood. We have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricles undergo restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs co-exist with monociliated cells and generate directional flow patterns. These ciliated cells have different developmental origins and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from the tela choroida and choroid plexus or global perturbation of multiciliation does not affect overall brain or spine morphogenesis but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.

Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments.

Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

PDCD4 controls the G1/S-phase transition in a telomerase-immortalized epithelial cell line and affects the expression level and translation of multiple mRNAs.

PDCD4, the protein encoded by the tumor suppressor gene PDCD4 (programmed cell death 4) has been implicated in the control of cellular transcription and translation by modulating the activity of specific transcription factors and suppressing the translation of mRNAs with structured 5'-UTRs. Most studies of human PDCD4 have employed tumor cell lines, possibly resulting in a biased picture of its role in normal cells. Here, we have studied the function of PDCD4 in a telomerase-immortalized human epithelial cell line. We show for the first time that PDCD4 is required for the G1/S-transition, demonstrating its crucial role in the cell cycle. Inhibition of p53-dependent activation of p21WAF1/CIP1 overrides the requirement for PDCD4 for the G1/S-transition, suggesting that PDCD4 counteracts basal p53 activity to prevent activation of the G1/S checkpoint by p53. Transcriptome and ribosome profiling data show that silencing of PDCD4 changes the expression levels and translation of many mRNAs, providing an unbiased view of the cellular processes that are affected by PDCD4 in an epithelial cell line. Our data identify PDCD4 as a key regulator of cell cycle- and DNA-related functions that are inhibited when it is silenced, suggesting that decreased expression of PDCD4 might contribute to tumor development by compromising genomic integrity.

The dual methyltransferase METTL13 targets N terminus and Lys55 of eEF1A and modulates codon-specific translation rates.

Eukaryotic elongation factor 1 alpha (eEF1A) delivers aminoacyl-tRNA to the ribosome and thereby plays a key role in protein synthesis. Human eEF1A is subject to extensive post-translational methylation, but several of the responsible enzymes remain unknown. Using a wide range of experimental approaches, we here show that human methyltransferase (MTase)-like protein 13 (METTL13) contains two distinct MTase domains targeting the N terminus and Lys55 of eEF1A, respectively. Our biochemical and structural analyses provide detailed mechanistic insights into recognition of the eEF1A N terminus by METTL13. Moreover, through ribosome profiling, we demonstrate that loss of METTL13 function alters translation dynamics and results in changed translation rates of specific codons. In summary, we here unravel the function of a human MTase, showing that it methylates eEF1A and modulates mRNA translation in a codon-specific manner.

Enzymatic or In Vivo Installation of Propargyl Groups in Combination with Click Chemistry for the Enrichment and Detection of Methyltransferase Target Sites in RNA.

m6 A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome-wide assignment of m6 A sites is of utmost importance. However, m6 A does not interfere with Watson-Crick base pairing, making polymerase-based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl groups can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m6 A sites after bioconjugation and purification, hence enabling detection of METTL3-METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl-l-selenohomocysteine and validated different types of known rRNA methylation sites.

Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation.

Many cellular proteins are methylated on lysine residues and this has been most intensively studied for histone proteins. Lysine methylations on non-histone proteins are also frequent, but in most cases the functional significance of the methylation event, as well as the identity of the responsible lysine (K) specific methyltransferase (KMT), remain unknown. Several recently discovered KMTs belong to the so-called seven-ß-strand (7BS) class of MTases and we have here investigated an uncharacterized human 7BS MTase currently annotated as part of the endothelin converting enzyme 2, but which should be considered a separate enzyme. Combining in vitro enzymology and analyzes of knockout cells, we demonstrate that this MTase efficiently methylates K36 in eukaryotic translation elongation factor 1 alpha (eEF1A) in vitro and in vivo. We suggest that this novel KMT is named eEF1A-KMT4 (gene name EEF1AKMT4), in agreement with the recently established nomenclature. Furthermore, by ribosome profiling we show that the absence of K36 methylation affects translation dynamics and changes translation speed of distinct codons. Finally, we show that eEF1A-KMT4 is part of a novel family of human KMTs, defined by a shared sequence motif in the active site and we demonstrate the importance of this motif for catalytic activity.

The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1 alpha (eEF1A).

Lysine methylation is abundant on histone proteins, representing a dynamic regulator of chromatin state and gene activity, but is also frequent on many non-histone proteins, including eukaryotic elongation factor 1 alpha (eEF1A). However, the functional significance of eEF1A methylation remains obscure and it has remained unclear whether eEF1A methylation is dynamic and subject to active regulation. We here demonstrate, using a wide range of in vitro and in vivo approaches, that the previously uncharacterized human methyltransferase METTL21B specifically targets Lys-165 in eEF1A in an aminoacyl-tRNA- and GTP-dependent manner. Interestingly, METTL21B-mediated eEF1A methylation showed strong variation across different tissues and cell lines, and was induced by altering growth conditions or by treatment with certain ER-stress-inducing drugs, concomitant with an increase in METTL21B gene expression. Moreover, genetic ablation of METTL21B function in mammalian cells caused substantial alterations in mRNA translation, as measured by ribosomal profiling. A non-canonical function for eEF1A in organization of the cellular cytoskeleton has been reported, and interestingly, METTL21B accumulated in centrosomes, in addition to the expected cytosolic localization. In summary, the present study identifies METTL21B as the enzyme responsible for methylation of eEF1A on Lys-165 and shows that this modification is dynamic, inducible and likely of regulatory importance.

Dual randomization of oligonucleotides to reduce the bias in ribosome-profiling libraries.

Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosome-profiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5' end and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3' end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation.