Interferon regulates neural stem cell function at all ages by orchestrating mTOR and cell cycle.

Stem cells show intrinsic interferon signalling, which protects them from viral infections at all ages. In the ageing brain, interferon signalling also reduces the ability of stem cells to activate. Whether these functions are linked and at what time interferons start taking on a role in stem cell functioning is unknown. Additionally, the molecular link between interferons and activation in neural stem cells and how this relates to progenitor production is not well understood. Here we combine single-cell transcriptomics, RiboSeq and mathematical models of interferon to show that this pathway is important for proper stem cell function at all ages in mice. Interferon orchestrates cell cycle and mTOR activity to post-transcriptionally repress Sox2 and induces quiescence. The interferon response then decreases in the subsequent maturation states. Mathematical simulations indicate that this regulation is beneficial for the young and harmful for the old brain. Our study establishes molecular mechanisms of interferon in stem cells and interferons as genuine regulators of stem cell homeostasis and a potential therapeutic target to repair the ageing brain.

Onset of differentiation is post-transcriptionally controlled in adult neural stem cells.

Whether post-transcriptional regulation of gene expression controls differentiation of stem cells for tissue renewal remains unknown. Quiescent stem cells exhibit a low level of protein synthesis1, which is key to maintaining the pool of fully functional stem cells, not only in the brain but also in the bone marrow and hair follicles2-6. Neurons also maintain a subset of messenger RNAs in a translationally silent state, which react 'on demand' to intracellular and extracellular signals. This uncoupling of general availability of mRNA from translation into protein facilitates immediate responses to environmental changes and avoids excess production of proteins, which is the most energy-consuming process within the cell. However, when post-transcriptional regulation is acquired and how protein synthesis changes along the different steps of maturation are not known. Here we show that protein synthesis undergoes highly dynamic changes when stem cells differentiate to neurons in vivo. Examination of individual transcripts using RiboTag mouse models reveals that whereas stem cells translate abundant transcripts with little discrimination, translation becomes increasingly regulated with the onset of differentiation. The generation of neurogenic progeny involves translational repression of a subset of mRNAs, including mRNAs that encode the stem cell identity factors SOX2 and PAX6, and components of the translation machinery, which are enriched in a pyrimidine-rich motif. The decrease of mTORC1 activity as stem cells exit the cell cycle selectively blocks translation of these transcripts. Our results reveal a control mechanism by which the cell cycle is coupled to post-transcriptional repression of key stem cell identity factors, thereby promoting exit from stemness.

An Immune-CNS Axis Activates Remote Hippocampal Stem Cells Following Spinal Transection Injury.

External stimuli such as injury, learning, or stress influence the production of neurons by neural stem cells (NSCs) in the adult mammalian brain. These external stimuli directly impact stem cell activity by influencing areas directly connected or in close proximity to the neurogenic niches of the adult brain. However, very little is known on how distant injuries affect NSC activation state. In this study, we demonstrate that a thoracic spinal transection injury activates the distally located hippocampal-NSCs. This activation leads to a transient increase production of neurons that functionally integrate to improve animal's performance in hippocampal-related memory tasks. We further show that interferon-CD95 signaling is required to promote injury-mediated activation of remote NSCs. Thus, we identify an immune-CNS axis responsible for injury-mediated activation of remotely located NSCs.

Neural Stem Cell Activation and the Role of Protein Synthesis.

Adult neural stem cells are generated at embryonic stages by entering a quiescent state that allows their retention into adulthood and thereby maintenance of life-long brain homeostasis. Thus, a tight balance between the quiescence and activation state is instrumental to meet the brain demands for a specific cell type at the correct numbers, at a given time and position. Protein synthesis is the most energy-consuming process within the cell and, not surprisingly, it occurs at low rates in quiescent stem cells. This way quiescent cells adjust to energy constraints and avoid their premature depletion. Stem cell activation is characterized by upregulation of protein synthesis followed by cell division and differentiation. The role of such upregulation as causative or rather a consequence of the activation remains elusive. Here we summarize recent findings connecting stem cell activation to the regulation of protein synthesis, particularly focusing on embryonic and adult neural stem cells of the ventricular zone.

5' UTR m(6)A Promotes Cap-Independent Translation.

Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5' cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent manner through poorly understood mechanisms. Here, we show that mRNAs containing N(6)-methyladenosine (m(6)A) in their 5' UTR can be translated in a cap-independent manner. A single 5' UTR m(6)A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5'UTR m(6)A. Additionally, increased m(6)A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m(6)A, resulting in increased numbers of mRNAs with 5' UTR m(6)A. These data show that 5' UTR m(6)A bypasses 5' cap-binding proteins to promote translation under stresses.

Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES.

The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP ⋅ eRF1 ⋅ eRF3 ⋅ GMPPNP and 80S⋅CrPV-STOP ⋅ eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1 ⋅ eRF3 ⋅ GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling.

Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs.

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5'-terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate (ppp) bridge. Cap0 is further modified by 2'-O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) and 'cap2' (m7GpppNmNm). However, some viral RNAs lack 2'-O-methylation, whereas others contain only ppp- at their 5'-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5'ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2'-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5'-terminal regions of 5'ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5'-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5'ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.

Reinitiation and other unconventional posttermination events during eukaryotic translation.

During ribosome recycling, posttermination complexes are dissociated by ABCE1 and eRF1 into 60S and tRNA/mRNA-associated 40S subunits, after which tRNA and mRNA are released by eIF1/eIF1A, Ligatin, or MCT-1/DENR. Occasionally, 40S subunits remain associated with mRNA and reinitiate at nearby AUGs. We recapitulated reinitiation using a reconstituted mammalian translation system. The presence of eIF2, eIF3, eIF1, eIF1A, and Met-tRNAi(Met) was sufficient for recycled 40S subunits to remain on mRNA, scan bidirectionally, and reinitiate at upstream and downstream AUGs if mRNA regions flanking the stop codon were unstructured. Imposition of 3' directionality additionally required eIF4F. Strikingly, posttermination ribosomes were not stably anchored on mRNA and migrated bidirectionally to codons cognate to the P site tRNA. Migration depended on the mode of peptide release (puromycin > eRF1⋅eRF3) and nature of tRNA and was enhanced by eEF2. The mobility of posttermination ribosomes suggests that some reinitiation events could involve 80S ribosomes rather than 40S subunits.

Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes.

No-go decay (NGD) and non-stop decay (NSD) are eukaryotic surveillance mechanisms that target mRNAs on which elongation complexes (ECs) are stalled by, for example, stable secondary structures (NGD) or due to the absence of a stop codon (NSD). Two interacting proteins Dom34(yeast)/Pelota(mammals) and Hbs1, which are paralogues of eRF1 and eRF3, are implicated in these processes. Dom34/Hbs1 were shown to promote dissociation of stalled ECs and release of intact peptidyl-tRNA. Using an in vitro reconstitution approach, we investigated the activities of mammalian Pelota/Hbs1 and report that Pelota/Hbs1 also induced dissociation of ECs and release of peptidyl-tRNA, but only in the presence of ABCE1. Whereas Pelota and ABCE1 were essential, Hbs1 had a stimulatory effect. Importantly, ABCE1/Pelota/Hbs1 dissociated ECs containing only a limited number of mRNA nucleotides downstream of the P-site, which suggests that ABCE1/Pelota/Hbs1 would disassemble NSD complexes stalled at the 3'-end, but not pre-cleavage NGD complexes stalled in the middle of mRNA. ABCE1/Pelota/Hbs1 also dissociated vacant 80S ribosomes, which stimulated 48S complex formation, suggesting that Pelota/Hbs1 have an additional role outside of NGD.

Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling.

Eukaryotic translation initiation begins with ribosomal recruitment of aminoacylated initiator tRNA (Met-tRNA(Met)(i)) by eukaryotic initiation factor eIF2. In cooperation with eIF3, eIF1, and eIF1A, Met-tRNA(Met)(i)/eIF2/GTP binds to 40S subunits yielding 43S preinitiation complexes that attach to the 5'-terminal region of mRNAs and then scan to the initiation codon to form 48S initiation complexes with established codon-anticodon base-pairing. Stress-activated phosphorylation of eIF2alpha reduces the level of active eIF2, globally inhibiting translation. However, translation of several viral mRNAs, including Sindbis virus (SV) 26S mRNA and mRNAs containing hepatitis C virus (HCV)-like IRESs, is wholly or partially resistant to inhibition by eIF2 phosphorylation, despite requiring Met-tRNA(Met)(i). Here we report the identification of related proteins that individually (Ligatin) or together (the oncogene MCT-1 and DENR, which are homologous to N-terminal and C-terminal regions of Ligatin, respectively) promote efficient eIF2-independent recruitment of Met-tRNA(Met)(i) to 40S/mRNA complexes, if attachment of 40S subunits to the mRNA places the initiation codon directly in the P site, as on HCV-like IRESs and, as we show here, SV 26S mRNA. In addition to their role in initiation, Ligatin and MCT-1/DENR can promote release of deacylated tRNA and mRNA from recycled 40S subunits after ABCE1-mediated dissociation of post-termination ribosomes.

The role of ABCE1 in eukaryotic posttermination ribosomal recycling.

After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA, and release factor eRF1 and must be recycled by dissociating these ligands and separating ribosomes into subunits. Although recycling of eukaryotic posttermination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1, and eIF1A (Pisarev et al., 2007), this energy-free mechanism can function only in a narrow range of low Mg(2+) concentrations. Here, we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg(2+) concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP, and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1's absence.

General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation.

The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.

Proteasome-mediated cleavage of the Y-box-binding protein 1 is linked to DNA-damage stress response.

YB-1 is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA- and RNA-dependent events is determined by its localization in the cell. Distribution of YB-1 between the nucleus and the cytoplasm is known to be dependent on nuclear targeting and cytoplasmic retention signals located within the C-terminal portion of YB-1. Here, we report that YB-1 undergoes a specific proteolytic cleavage by the 20S proteasome, which splits off the C-terminal 105-amino-acid-long YB-1 fragment containing a cytoplasmic retention signal. Cleavage of YB-1 by the 20S proteasome in vitro appears to be ubiquitin- and ATP-independent, and is abolished by the association of YB-1 with messenger RNA. We also found that genotoxic stress triggers a proteasome-mediated cleavage of YB-1 in vivo and leads to accumulation of the truncated protein in nuclei of stressed cells. Endoproteolytic activity of the proteasome may therefore play an important role in regulating YB-1 functioning, especially under certain stress conditions.

YB-1 autoregulates translation of its own mRNA at or prior to the step of 40S ribosomal subunit joining.

YB-1 is a member of the numerous families of proteins with an evolutionary ancient cold-shock domain. It is involved in many DNA- and RNA-dependent events and regulates gene expression at different levels. Previously, we found a regulatory element within the 3' untranslated region (UTR) of YB-1 mRNA that specifically interacted with YB-1 and poly(A)-binding protein (PABP); we also showed that PABP positively affected YB-1 mRNA translation in a poly(A) tail-independent manner (O. V. Skabkina, M. A. Skabkin, N. V. Popova, D. N. Lyabin, L. O. Penalva, and L. P. Ovchinnikov, J. Biol. Chem. 278:18191-18198, 2003). Here, YB-1 is shown to strongly and specifically inhibit its own synthesis at the stage of initiation, with accumulation of its mRNA in the form of free mRNPs. YB-1 and PABP binding sites have been mapped on the YB-1 mRNA regulatory element. These were UCCAG/ACAA for YB-1 and a approximately 50-nucleotide A-rich sequence for PABP that overlapped each other. PABP competes with YB-1 for binding to the YB-1 mRNA regulatory element and restores translational activity of YB-1 mRNA that has been inhibited by YB-1. Thus, YB-1 negatively regulates its own synthesis, presumably by specific interaction with the 3'UTR regulatory element, whereas PABP restores translational activity of YB-1 mRNA by displacing YB-1 from this element.

Structural organization of mRNA complexes with major core mRNP protein YB-1.

YB-1 is a universal major protein of cytoplasmic mRNPs, a member of the family of multifunctional cold shock domain proteins (CSD proteins). Depending on its amount on mRNA, YB-1 stimulates or inhibits mRNA translation. In this study, we have analyzed complexes formed in vitro at various YB-1 to mRNA ratios, including those typical for polysomal (translatable) and free (untranslatable) mRNPs. We have shown that at mRNA saturation with YB-1, this protein alone is sufficient to form mRNPs with the protein/RNA ratio and the sedimentation coefficient typical for natural mRNPs. These complexes are dynamic structures in which the protein can easily migrate from one mRNA molecule to another. Biochemical studies combined with atomic force microscopy and electron microscopy showed that mRNA-YB-1 complexes with a low YB-1/mRNA ratio typical for polysomal mRNPs are incompact; there, YB-1 binds to mRNA as a monomer with its both RNA-binding domains. At a high YB-1/mRNA ratio typical for untranslatable mRNPs, mRNA-bound YB-1 forms multimeric protein complexes where YB-1 binds to mRNA predominantly with its N-terminal part. A multimeric YB-1 comprises about twenty monomeric subunits; its molecular mass is about 700 kDa, and it packs a 600-700 nt mRNA segment on its surface.

Poly(A)-binding protein positively affects YB-1 mRNA translation through specific interaction with YB-1 mRNA.

The major protein of cytoplasmic mRNPs from rabbit reticulocytes, YB-1, is a member of an ancient family of proteins containing a common structural feature, cold-shock domain. In eukaryotes, this family is represented by multifunctional mRNA/Y-box DNA-binding proteins that control gene expression at different stages. To address possible post-transcriptional regulation of YB-1 gene expression, we examined effects of exogenous 5'- and 3'-untranslatable region-containing fragments of YB-1 mRNA on its translation and stability in a cell-free system. The addition of the 3' mRNA fragment as well as its subfragment I shut off protein synthesis at the initiation stage without affecting mRNA stability. UV cross-linking revealed four proteins (69, 50, 46, and 44 kDa) that specifically interacted with the 3' mRNA fragment; the inhibitory subfragment I bound two of them, 69- and 50-kDa proteins. We have identified these proteins as PABP (poly(A)-binding protein) (69 kDa) and YB-1 (50 kDa) and demonstrated that titrating out of PABP by poly(A) strongly and specifically inhibits YB-1 mRNA cap(+)poly(A)(-) translation in a cell-free system. Thus, PABP is capable of positively affecting YB-1 mRNA translation in a poly(A) tail-independent manner.