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Protein methylation was discovered over 50 years ago, but only with the advent of genomic and proteomic technologies could its mechanisms and cellular functions be studied in detail. Shi and Murn discuss the seminal discoveries in protein methylation research and highlight future directions for this field. In 1959, while analysing the bacterial flagellar proteins, Ambler and Rees observed an unknown species of amino acid that they eventually identified as methylated lysine. Over half a century later, protein methylation is known to have a regulatory role in many essential cellular processes that range from gene transcription to signal transduction. However, the road to this now burgeoning research field was obstacle-ridden, not least because of the inconspicuous nature of the methyl mark itself. Here, we chronicle the milestone achievements and discuss the future of protein methylation research.

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs—mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)—that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation. Shi et al. profiled small non-coding RNAs (sncRNAs) through PANDORA-seq, which identified tissue-specific transfer RNA- and ribosomal RNA-derived small RNAs, as well as sncRNAs, with dynamic changes during induced pluripotent stem cell reprogramming.

Cell fate commitment is driven by dynamic changes in chromatin architecture and activity of lineage-specific transcription factors (TFs). The chromatin assembly factor-1 (CAF-1) is a histone chaperone that regulates chromatin architecture by facilitating nucleosome assembly during DNA replication. Accumulating evidence supports a substantial role of CAF-1 in cell fate maintenance, but the mechanisms by which CAF-1 restricts lineage choice remain poorly understood. Here, we investigate how CAF-1 influences chromatin dynamics and TF activity during lineage differentiation. We show that CAF-1 suppression triggers rapid differentiation of myeloid stem and progenitor cells into a mixed lineage state. We find that CAF-1 sustains lineage fidelity by controlling chromatin accessibility at specific loci, and limiting the binding of ELF1 TF at newly-accessible diverging regulatory elements. Together, our findings decipher key traits of chromatin accessibility that sustain lineage integrity and point to a powerful strategy for dissecting transcriptional circuits central to cell fate commitment. Cell fate commitment involves transcription factor activity and changes in chromatin architecture. Here the authors show that CAF-1 maintains lineage fidelity by controlling chromatin accessibility at specific sites; suppressing CAF-1 triggers differentiation of myeloid stem and progenitor cells into a mixed lineage state.

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. RNA interference screens were used to identify chromatin-associated factors that impede reprogramming of somatic cells into iPS cells; suppression of the chromatin assembly factor CAF-1 enhances the generation of iPS cells by rendering chromatin more accessible to pluripotency transcription factors. CAF-1 is a barrier to cell fate change Lineage fate determination in development, and reprogramming to a different fate in the laboratory, depend on gene expression programs that are regulated by factors influencing the chromatin landscape. Konrad Hochedlinger and colleagues have performed two RNA-interference-based screens to search for chromatin-associated factors that impede reprogramming. They identify the chromatin assembly factor-1 (CAF-1) complex as an important regulator of this process, acting by rendering the chromatin inaccessible to transcription factors. Suppression of CAF-1 function thus facilitates chromatin access to reprogramming factors, both during reprogramming to pluripotency and in direct fate conversion.

How RNA-binding proteins (RBPs) convey regulatory instructions to the core effectors of RNA processing is unclear. Here, we document the existence and functions of a multivalent RBP–effector interface. We show that the effector interface of a conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of ‘dual-purpose’ peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by Unkempt. Systems analyses reveal that multivalent RBP–effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for the reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP–effector interface. How RNA-binding proteins (RBPs) regulate gene expression via effectors of RNA processing is unclear. Here, the authors dissect the effector interface of an essential RBP, Unkempt, and investigate its contribution to translational control in cells.

While evolution is often considered from a DNA- and protein-centric view, RNA-based regulation can also impact gene expression and protein sequences. Here we examine interspecies differences in RNA-protein interactions using the conserved neuronal RNA-binding protein, Unkempt (UNK) as model. We find that roughly half of mRNAs bound in human are also bound in mouse. Unexpectedly, even when transcript-level binding was conserved across species differential motif usage was prevalent. To understand the biochemical basis of UNK-RNA interactions, we reconstitute the human and mouse UNK-RNA interactomes using a high-throughput biochemical assay. We uncover detailed features driving binding, show that in vivo patterns are captured in vitro, find that highly conserved sites are the strongest bound, and associate binding strength with downstream regulation. Furthermore, subtle sequence differences surrounding motifs are key determinants of species-specific binding. We highlight the complex features driving protein-RNA interactions and how these evolve to confer species-specific regulation. This study details how RNA-binding sites evolved between human and mouse, focusing on the conserved neuronal protein, Unkempt. The authors uncover that RNAbinding proteins often change binding sites and regulation across species even when binding motifs are conserved.

Cellular quiescence is a state of reversible proliferative arrest that plays essential roles in development, resistance to stress, aging, and longevity of organisms. Here we report that rapid depletion of RNase MRP, a deeply conserved RNA-based enzyme required for rRNA biosynthesis, induces a long-term yet reversible proliferative arrest in human cells. Severely compromised biogenesis of rRNAs along with acute transcriptional reprogramming precede a gradual decline of the critical cellular functions. Unexpectedly, many arresting cells show increased levels of histone mRNAs, which accumulate locally in the cytoplasm, and S-phase DNA amount. The ensuing proliferative arrest is entered from multiple stages of the cell cycle and can last for several weeks with uncompromised cell viability. Strikingly, restoring expression of RNase MRP leads to a complete reversal of the arrested state with resumed cell proliferation at the speed of control cells. We suggest that targeting rRNA biogenesis may provide a general strategy for rapid induction of a reversible proliferative arrest, with implications for understanding and manipulating cellular quiescence. Cellular quiescence is a reversible dormant state that helps cells survive stress, support tissue regeneration, and promote longevity. Here, the authors show that blocking ribosomal RNA production by rapid depletion of RNase MRP induces a long-lasting quiescence-like state in human cells.

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human‐induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems‐level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. Synopsis Rapid and homogeneous neuronal differentiation is attained in human stem cells upon overexpression of two Neurogenin transcription factors. mRNA and miRNA expression profiling during differentiation reveals a regulatory network mediating neurogenesis from stem cells. Neurogenin‐1 and ‐2 drive homogeneous differentiation of human stem cells into bipolar neurons in 4 days in defined media. The population homogeneity allowed mRNA and miRNA expression profiling over time during neurogenesis. A network of key transcription factors and miRNAs that promote rapid neurogenesis and loss of pluripotency is identified. Perturbations of key transcription factors affect the homogeneity and phenotypic properties of the resulting neurons. Graphical Abstract Rapid and homogeneous neuronal differentiation is attained in human stem cells upon overexpression of two Neurogenin transcription factors. mRNA and miRNA expression profiling during differentiation reveals a regulatory network mediating neurogenesis from stem cells.

Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila . Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt ( Unk cKO ) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in Unk cKO mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that Unk cKO mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.

A new crystal structure of mouse Unkempt, a translational regulator of neuronal cell morphology, reveals how its two zinc-finger-triplet clusters recognize distinct cognate RNA sites. Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. Here we determined the X-ray crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, ZnF1–3 and ZnF4–6, that recognize distinct trinucleotide RNA substrates. Both ZnF clusters adopt a similar overall topology and use distinct recognition principles to target specific RNA sequences. Structure-guided point mutations reduce the RNA binding affinity of Unkempt both in vitro and in vivo , ablate Unkempt's translational control and impair the ability of Unkempt to induce a bipolar cellular morphology. Our study unravels a new mode of RNA sequence recognition by clusters of CCCH ZnFs that is critical for post-transcriptional control of neuronal morphology.