In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion. Summary: The short isoform of TOM-1/tomosyn inhibits growth cone protrusion downstream of the netrin receptor UNC-5 during growth cone outgrowth, whereas long isoforms have a pro-protrusive role. Guided extensions of axons and dendrites play a crucial role in the development of neural circuits and networks. Growth cones present at the growing tip of the neurite guide the outgrowth of the neurite. Growth cones are dynamic structures comprising a branched actin lamellipodial body and filopodial protrusions consisting of F-actin bundles . ExtraceCaenorhabditis elegans and vertebrates, UNC-6/netrin is a bifunctional, conserved, secreted laminin-like guidance cue, which directs dorsal-ventral axon guidance by utilizing its receptors UNC-40/DCC and UNC-5  was identified as a syntaxin-interacting molecule that was shown to inhibit interaction of the T-SNARE syntaxin with the V-SNARE synaptobrevin, thus blocking formation of the SNARE complex and preventing vesicle fusion . Tomosyn elegans . Tomosyn elegans . When gr elegans .tom-1 encodes long (TOM-1L) and short (TOM-1S) isoforms. The long isoform contains N-terminal WD40 repeats and a C-terminal R-SNARE domain that interacts with syntaxin, whereas the short isoform only encodes the C-terminal R-SNARE domain and lacks the WD40 repeats. A mutation that eliminates only the long isoform but not the short isoform resulted in small, less-protrusive VD growth cones. Furthermore, tom-1L mutation suppressed the excess VD growth cone protrusion observed in unc-5 loss-of-function mutants. These results suggest that the TOM-1L and TOM-1S isoforms have opposing functions, with TOM-1S acting in an anti-protrusive manner and TOM-1L being pro-protrusive. Mutation of tom-1S without affecting tom-1L led to a phenotype resembling that of complete loss of tom-1 . Furthermore, transgenic expression of tom-1S resulted in reduced VD growth cone protrusion. Taken together, these data suggest that in VD growth cones tom-1S is the active isoform in inhibiting growth cone protrusion and possibly vesicle fusion, whereas tom-1L has a pro-protrusive role. unc-64 encodes the T-SNARE syntaxin. A hypomorphic unc-64 mutant had small, less-protrusive VD growth cones and suppressed the excess protrusion of unc-5 loss-of-function mutants. In sum, these results are consistent with a model wherein UNC-5 engages TOM-1S to inhibit vesicle fusion and thus inhibit growth cone protrusion. Additionally, tom-1L, tom-1S and unc-64 were each required for VD growth cone polarity of protrusion, suggesting that regulated vesicle fusion is necessary to establish and/or maintain VD growth cone polarity.In this work, the role of TOM-1 in VD growth cone morphology is explored as well as its interaction with UNC-5 in inhibiting VD growth cone protrusion. Although complete loss of TOM-1 had little effect on VD growth cone morphology, it did suppress growth cone inhibition of protrusion driven by activated MYR::UNC-5. This suggests that TOM-1 is required for MYR::UNC-5 to inhibit growth cone protrusion and that TOM-1 might act downstream of UNC-5 in this process. C. elegans genome encodes a single tomosyn gene, tom-1, which was identified in a forward genetic screen for enhancers of acetylcholine secretion (Stxbp5) and tomosyn 2 (Stxbp5l) (tom-1 produces at least seven isoforms (WormBase): six long isoforms, including tom-1A, and a single short isoform encoded by tom-1B (tom-1A. RNA sequencing (RNA-seq) on three independent replicates of mixed-stage, wild-type animals revealed that the tom-1B 5′ exon and splice occurred in 8/137, 9/151 and 4/109, or ∼5%, of splicing events involving this intron  . Throughy tom-1B A, producs intron B. This i complex A (Grache complex . The shotom-1(ok2437) is a 2391 bp deletion that removes 3′ exons predicted to affect the short and long isoforms of tom-1 (tom-1(nu468) introduces a premature stop codon at tryptophan at 212  and tom-1(ok2437) mutants each displayed increased levels of axon guidance defects compared with wild type. However, these increases were not statistically significant , an allele affecting both long and short isoforms, displayed a slight but not statistically significant increase in VD growth cone area and filopodial length (tom-1(nu468) mutant VD growth cones displayed significantly reduced growth cone area and filopodial length  and tom-1(nu468) mutants (tom-1(nu468) mutant displayed reduced growth cone area and shorter filopodial protrusions, whereas the tom-1(ok2437) mutant, in which both long and short isoforms are affected, did not. This suggests that the long and short isoforms of TOM-1 might have distinct roles in regulation of VD growth cone morphology.Dorsal polarity of growth cone protrusion was significantly reduced in both  mutants C,E,F. ThPrevious work showed that UNC-6/netrin signaling regulates VD growth cone protrusion, including growth cone area, filopodial length, and polarity . The UNCtom-1(ok2437), which affects both long and short isoforms, significantly suppressed the effects of myr::unc-5. tom-1(ok2437); myr::unc-5 growth cones were larger with longer filopodia compared with myr::unc-5 alone (tom-1(ok2437) had no effect on growth cone area or filopodial reduction caused by myr::unc-40 , which affects only the tom-1L isoforms and not the tom-1S isoform, did not suppress the inhibition of growth cone area and filopodial length caused by myr::unc-5 or myr::unc-40 (tom-1(nu468) mutants displayed reduced VD growth cone protrusion (tom-1(ok2437), tom-1(nu468), myr::unc-5 and myr::unc-40 each affected growth cone polarity, and polarity was unchanged in double mutants , axon guidance defects were increased. This suggests that growth cone protrusion is not the only aspect of growth cone dynamics during axon guidance affected by these molecules. did not E-H, 3, cunc-5(e791) null mutants displayed increased growth cone area and filopodial length (unc-5(ev480) and unc-5(e152) are hypomorphic alleles that retain some UNC-5 function (unc-5(ev480) and unc-5(e152) displayed VD growth cone morphology similar to the unc-5(e791) null  mutants displayed a nearly complete failure of VD/DD axons to reach the dorsal nerve cord, whereas unc-5(ev480) and unc-5(e152) displayed weaker axon guidance defects owing to their hypomorphic nature  null A,B,D,G. trusions C. unc-5(c nature .tom-1(ok2437) null allele and the unc-5(e791) null allele could not be scored because none emerged from the ventral nerve cord. There was a resulting complete failure of VD or DD axons extending out of the ventral nerve cord (tom-1(ok2437) with hypomorphic unc-5(e152) and unc-5(ev480) were apparent, and they resembled the growth cones of unc-5 hypomorphs alone, with increased growth cone area and filopodial length compared with wild type  alone were observed (tom-1(ok2437) did not alter the growth cone phenotypes of unc-5 hypomorphs, and in fact enhanced VD/DD axon guidance defects.VD growth cones of double mutants of the rve cord M,N. VD gild type A,B,E. Altom-1ok247 null altom-1(nu468) mutant elicited significantly suppressed growth cone area and filopodial length in all unc-5 mutants  allele is predicted to encode the long isoform of TOM-1.The  isoform A. We usese exons . This totom-1(lq176) did not affect growth cone area and filopodial length compared with wild type (tom-1(lq176) suppressed the inhibition of growth cone area and filopodial length caused by myr::unc-5 (unc-5(ev480 and e152) mutants (tom-1(lq176); unc-5(e791) null double mutants. All double mutants with tom-1(lq176), unc-5 hypomorphs, and myr::unc-5 displayed slightly increased VD/DD axon guidance defects, which were in some cases statistically significant (tom-1(lq176) also displayed increased levels of axon guidance defects relative to wild type, but this was not statistically significant.ild type A,B,D,E, ild type C. tom-1 resembled tom-1(ok2437) in all respects, including loss of growth cone polarity, suppression of growth cone inhibition by activated myr::unc-5, and failure to suppress excess growth cone protrusion in unc-5 hypomorphic mutants. This indicates that the TOM-1 long isoforms alone cannot supply full TOM-1 function and is consistent with long and short isoforms having opposing roles .In sum, tom-1S short isoform was placed under the control of the unc-25 promoter expressed in the VD/DD neurons. Transgenic expression of this construct in a wild-type background resulted in reduced VD growth cone area and filopodial length , an allele affecting both long and short isoforms (tom-1(lq176). These data also indicate that tom-1S can act cell-autonomously to inhibit growth cone protrusion. To investigate further whether TOM-1S isoform acts downstream of UNC-5, we analyzed double mutants of Punc-25::tom-1S short and unc-5. Double mutants displayed significant suppression of growth cone area and filopodial length compared with single unc-5 mutants  and unc-5(e791) mutants , was placed under the control of the unc-25 promoter expressed in the VD/DD neurons. Three independent transgenic lines were scored. Two showed no significant change to growth cone morphology, and one displayed a decrease in the growth cone area and filopodial length (tom-1(ok2437) background. All three lines displayed a significant decrease in the dorsal polarity of protrusions  allele is viable and displays slightly uncoordinated locomotion  mutants displayed VD growth cones with reduced area and filopodial length compared with wild type (unc-64(md130) growth cones were unpolarized (unc-64(md130) suppressed excessive growth cone protrusion in unc-5 null and hypomorphic mutants (unc-64(md130) displayed weak axon guidance defects alone, but double mutants of unc-64; unc-5 displayed a synergistic enhancement in VD/DD axon guidance , UNC-6 directs dorsal migration of the VD growth cone. Previous studies showed that UNC-5 inhibits growth cone protrusion using FMOs, which might oxidize and destabilize F-actin in a similar manner to MICAL. UNC-5 also inhibits protrusion by restricting entry of microtubule plus-ends into growth cones, which have a pro-protrusive effect. Results here suggest that UNC-5 inhibits protrusion via a third pathway involving UNC-64 and the syntaxin inhibitor TOM-1  and thatin vivo results presented here are consistent with the idea that TOM-1 restricts vesicle fusion in the growth cone.Vesicle exocytosis is essential for controlling growth cone membrane dynamics and extensions by fusion of plasmalemmal precursor vesicles at the plasma membrane of growth cones . StudiesC. elegans genome contains one tomosyn gene, tom-1, which encodes N-terminal WD40 repeats and a C-terminal R-SNARE domain, similar to tomosyn in other species. tom-1 produces multiple isoforms, including a series of long isoforms containing the WD40 repeats and the R-SNARE domain, and a short isoform containing only the R-SNARE domain, produced by an alternative 5′ exon. This short isoform has not been described in Drosophila or vertebrates, but our results here suggest that it is an active isoform of TOM-1. Genetic results here indicate that TOM-1L isoforms have pro-protrusive roles in the growth cone, whereas TOM-1S inhibits protrusion. The short-isoform-specific tom-1(lq176) mutant resembled tom-1(ok2437), suggesting that the long isoform cannot provide TOM-1 function in the absence of the short isoform. These data suggest that the TOM-1S isoform is important for inhibiting growth cone protrusion, possibly through interaction with UNC-64. Transgenic expression of TOM-1S resulted in smaller growth cones with shorter filopodia, consistent with this model. TOM-1L might have pro-protrusive roles independent of the short isoform. Alternately, TOM-1L might regulate TOM-1S and inhibit its function  . We alsotom-1(ok2437) an allele affecting both the short and long isoforms and the short-isoform-specific tom-1(lq176) both suppressed the inhibitory effect of MYR::UNC-5 on VD growth cone protrusion. Although neither mutant alone had significantly increased growth protrusion expected of an inhibitory molecule, the effects might be masked by the other pathways downstream of UNC-5 (FMOs and UNC-33/CRMP), or by another redundant pathway. MYR::UNC-5 is a sensitized genetic background that revealed the effects on tom-1 mutants on the VD growth cone. tom-1(ok2437) and tom-1(lq176) did not suppress the inhibitory effects on MYR::UNC-40, suggesting that TOM-1 might act specifically downstream of UNC-5 homodimers and not UNC-5:UNC-40 heterodimers. Finally, tom-1(ok2437) and tom-1(lq176) did not affect the large, overly protrusive growth cones of unc-5 loss-of-function mutants, consistent with a role in inhibiting protrusion.Here we show that the TOM-1S isoform is required for UNC-5 to inhibit VD growth cone protrusion. The tom-1(ok2437) which affects both long and short isoforms and tom-1(lq176) short isoform specific mutant, the long isoform-specific mutant tom-1(nu468) displayed VD growth cones with reduced area and filopodial length. tom-1(nu468) was required for the large, overly protrusive growth cones in unc-5 null mutants, and did not suppress MYR::UNC-5. Together, these data indicate that TOM-1 long isoforms have a pro-protrusive role in the growth cone, the opposite of TOM-1 short. TOM-1L isoforms were required for excess protrusion in unc-5 mutants, suggesting that, in the absence of UNC-5, TOM-1L is overactive.In contrast to the Previous neurophysiological studies indicate that the TOM-1 long isoforms are inhibitory to neurosecretion . Expresstom-1 mutants analyzed here displayed loss of dorsally polarized filopodial protrusions on the growth cone. Transgenic expression of the TOM-1S also resulted in VD growth cone polarity defects. This indicates that both TOM-1L and TOM-1S are required to establish and/or maintain VD growth cone polarity in a complex and likely dynamic manner. No genetic interaction analyzed here modified the polarity defect, so it is impossible to say whether TOM-1 acts downstream of UNC-5 in growth cone polarity.All tom-1 alleles displayed increased VD/DD axon guidance defects , and all of them synergistically enhanced the VD/DD axon guidance defects in double mutant combinations with unc-5. This suggests that both isoforms of tom-1 are necessary to maintain proper axon guidance and again supports the hypothesis that TOM-1 acts downstream of UNC-5 signaling.All C. elegans TOM-1 in neurosecretion  mutant and is consistent with a role of UNC-64/syntaxin in promoting growth cone protrusion and polarity. This strongly indicates that vesicle fusion is required for robust VD growth cone protrusion, filopodial protrusion, and polarity of filopodial protrusion. The effects of TOM-1 on these processes are thus likely to be mediated through regulation of vesicle fusion.Tomosyn is very well-characterized as an inhibitor of vesicle fusion by blocking interaction of syntaxin with the V-SNARE synaptobrevin, including ecretion . It is pin vivo in C. elegans that TOM-1 might act similarly in the VD growth cone. Localization of TOM-1S puncta to the ventral region of the VD growth cone suggests that TOM-1S might be active at the base of the VD growth cone. Loss of UNC-64 also resulted reduced growth cone protrusion. We speculate that TOM-1S prevents vesicle fusion ventrally, resulting in inhibited ventral growth cone protrusion.In cultured rat hippocampal neurons, tomosyn prevents vesicle fusion at the ‘palm’ of the growth cone, directing vesicle fusion to the extending growth cone tip . EvidencWe show that TOM-1 acts downstream of the UNC-6 receptor UNC-5 to inhibit protrusion. However, TOM-1S localization ventrally was not dependent on UNC-5A, suggesting that UNC-5 might activate TOM-1S and some other mechanism results in ventral localization of TOM-1S. Given that UNC-5 also polarizes the growth cone, the activity of TOM-1 ventrally and laterally might be controlled by UNC-5. TOM-1 is also required to establish and/or maintain growth cone polarity of protrusion, suggesting the role of vesicle fusion in this process as well. In cultured rat hippocampal neurons, upon growth cone collapse, tomosyn extends throughout the growth cone. This situation might be analogous to constitutively active MYR::UNC-5, which might constitutively recruit TOM-1 throughout the entire growth cone, leading to inhibited protrusion. These results advance our understanding of the role of the UNC-6 receptor UNC-5 in growth cone morphology during outgrowth. They show that, in addition to the two pathways involving F-actin and microtubule plus-end entry, UNC-5 inhibits protrusion by preventing vesicle fusion in the growth cone using TOM-1.C. elegans techniques , wrdSi23. LGII: juIs76 [Punc-25::gfp]. LGIII: unc-64(md130). LGIV: unc-5. The presence of mutations was confirmed by phenotype and sequencing. Chromosomal locations not determined: lqIs296[Punc-25::myr::unc-5::gfp], lqIs128[Punc-25::myr::unc-40::gfp], lqIs383 [Punc-25::tom-1S::gfp], lqIs346, lqIs347, lqIs382 .Experiments were performed at 20°C using standard chniques . MutatioPunc-25::tom-1S  and placed downstream of the unc-25 promoter to drive expression in the VD/DD neurons. The coding regions for both transgenes were sequenced to ensure no errors had been introduced by PCR.pEL1147;  was madetom-1A, between exons 18 and 19 (tom-1B short isoform (tom-1S), which resides in intron 18, and leaves the coding potential of tom-1 long isoforms unchanged. Synthetic guide RNAs were directed at the 5′ and 3′ ends of the intron: sgRNA1 against tom-1 short: 5′ CATCAATTTCCACAGAATGT 3′; sgRNA2 against tom-1 short: 5′ TTACATGGCAAGTCAAACAG 3′.CRISPR-Cas9 genome editing was used to delete in a precise manner the entire intron 18 of 8 and 19 . This redpy-10(cn64) co-CRISPR reagents . Genome-editing reagents were produced by InVivo Biosystems.A mix of sgRNAs, a single-stranded oligonucleotide repair template, and Cas9 enzyme was injected into the gonads of N2 animals, along with the reagents . DeletiowrdSi23 I; juIs76 II and is wild type for the unc-5 gene. Stranded poly-A RNA-seq libraries were constructed using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina and subjected to paired-end 150 cycle sequencing on a Nextseq550. Reads were aligned to the C. elegans genome using HISAT2 with default settings (version 2.1.0)  as previously described . The LE6n 2.1.0) . The resn 2.1.0)  from whit-test with unequal variance. With an α false-positive value of 0.05, sample sizes used each gave an estimated statistical power of >80% .Proportional data were analyzed using Fisher's Exact test, and continuous data with unpaired, two-tailed Student's Punc-25::gfp transgene juIs76  was much lower than 0.2.VD growth cones were imaged as previously described . Late L1t-test with unequal variance.Growth cones were imaged with a Qimaging Retiga EXi camera on a Leica DM5500 microscope at 100× magnification. Projections<0.5 μm in width were scored as filopodia. Growth cone area and filopodial length were quantified using ImageJ software. Quantification was performed as described previously . SignifiPolarity of growth filopodial protrusions was determined as previously described . Growth 10.1242/develop.201031_sup1Supplementary informationClick here for additional data file.


N-glycosyl derivatives of β-d-ribose. These N-ribosides are involved in most metabolic processes that occur in cells. They are essential components of nucleic acids, forming the basis for genetic information storage and flow. Moreover, these compounds are involved in numerous catalytic processes, including chemical energy production and storage, in which they serve as cofactors or coribozymes. From a chemical point of view, the overall structure of nucleotides and nucleosides is very similar and simple. However, their unique chemical and structural features render these compounds versatile building blocks that are crucial for life processes in all known organisms. Notably, the universal function of these compounds in encoding genetic information and cellular catalysis strongly suggests their essential role in the origins of life. In this review, we summarize major issues related to the role of N-ribosides in biological systems, especially in the context of the origin of life and its further evolution, through the RNA-based World(s), toward the life we observe today. We also discuss possible reasons why life has arisen from derivatives of β-d-ribofuranose instead of compounds based on other sugar moieties.Most naturally occurring nucleotides and nucleosides are  It is a challenge to define life based on the Natural Sciences in a simple yet exhaustive way ,2. AmongN-glycosidic derivatives of β-d-ribofuranose. Secondly, elements based on these N-glycosides provide genetic information as well as enable catalytic processes to occur. Indeed, a number of those processes based on, among others, the N-ribosides are a driving force for the self-sustainability of living chemical systems. Additionally, the genetic information is not copied and transcribed perfectly, especially that encoded in RNA carriers, and, thus, is capable of Darwinian evolution. Interestingly, a hypothesis proposed by Yarus [N-glycosidic derivatives of β-d-ribofuranoses in the origin of primary metabolic processes.The processes occurring in living organisms are highly complex and diverse. On the other hand, all currently known organisms function based on the central dogma of molecular biology, which determines the direction of information flow from the DNA through the RNA to proteins  and phosphate groups. An essential determinant was the presence of particular synthetic routes, which ensured access to specific chemical compounds in a prebiotic environment. However, conditions present on the Earth about four billion years ago presumably promoted numerous chemical reactions; thus, an environment contained various organic and inorganic compounds [d-ribonucleotides based on adenine, cytosine, guanine and uracil, which ensured the success of the “RNA-based World”. An interesting point is the homochirality of the sugar moiety in the “RNA-based World” [l-ribofuranose derivatives agrees well with the stereochemistry of experimental models of naturally occurring nucleic acids [d-amino acids and β-l-ribonucleotides, respectively), as revealed recently for T7 transcription [d-ribonucleotides instead of their l-enantiomers. However, some structural studies of RNA enantiomers have revealed differences on the RNA–solvent interfaces [Widely accepted hypotheses to explain that the origin of life includes the stage of the “RNA-based World” ,25,26,27ompounds . Therefoompounds ,31,32,33d World” , which cic acids . Moreovecription . It is uterfaces ,38. Howeterfaces .N-glycosyl derivatives of β-d-ribofuranose could be preferred over other derivatives. In a water solution at standard conditions, d-ribose exists in an equilibrium of four cyclic (five- or six-membered) hemiacetal forms, namely, α- and β-d-ribofuranose and α- and β-d-ribopyranose, and the latter forms are more stable (a relative abundance of six-membered forms is approximately 80% and only approximately 13% for β-d-ribofuranose) [N-ribosides is elevated [d-ribonucleotides over nucleotides based on glycerol [A definitive answer to why nature chose no other sugar moiety  but ribose as building blocks in the initial steps of abiogenesis and the origin of live is elusive. Herein, a severe obstacle is a lack of knowledge of the exact chemistry and physics in a prebiotic environment, which surely defined and promoted particular synthetic pathways as well as their stereoselectivity. However, some structural, chemical and biological premises suggest why uranose) . On the elevated . Importaelevated . A polynelevated ,44. Moreelevated ,33. In pglycerol ,30.d-ribonucleotides that were available as a result of the geochemical processes of that time [d-ribonucleotides containing four nucleobases, namely, cytosine, uracil, adenine and guanine, which are able to combine and form single RNA chains under appropriate conditions, is definitely more common nowadays [d-ribonucleotide synthesis under the prebiotic conditions of the time [d-nucleoside synthesis” [d-nucleotides in the prebiotic world. With the emergence of the “RNA World” theory, it was assumed the N-glycosidic bond was formed between the d-ribose and nitrogenous base molecules during the synthesis of β-d-ribonucleotides. However, this chemical pathway seems unlikely. The primary reason for that is the fact that, even if the d-ribose was produced in large quantities under prebiotic conditions, the chemical synthesis of β-d-nucleotides by using d-ribose and nitrogen heterocyclic bases as substrates is either very inefficient [d-ribose and a free base [N-glycosidic derivatives, in which both the sugar residue and the base are formed simultaneously on an activated precursor molecule containing a C-N bond of the emerging N-glycoside. This pathway of the synthesis of activated ribonucleotides, which are necessary for RNA synthesis, has another two advantages. The phosphate ions participating in the reaction are not only a substrate and a buffer but also serve as a catalyst. Additionally, such a pathway limits the variety of products at the initial stages of the reaction. A point of weakness of those chemical routes in the context of the “prebiotic chemist’s nightmare” problem is that syntheses are not selective, and products are derived from four aldopentose isomers. Moreover, the synthesis efficiency of d-ribosides is lower than that of other pentosides. The above limitations were overcome by Kim and Benner [N9-regioselective. Finally, Becker et al. [The “RNA World”, the hypothesis formulated by Gilbert , which ahat time ,60,61,62nowadays ,64,65. Ithe time ,62,66. Tnthesis” . The firfficient   into cellular-type lipid systems , which took place either simultaneously or sometime later, could have marked the occurrence of primary life on Earth ,71,72,73The success of the “RNA-based World(s)” most likely relied on an ability to self-replicate. Although a self-replicating single RNA molecule remains undiscovered , other rIt is of note that the aminoacylated RNA primer significantly enhances non-enzymatic primer extension and ligation reactions . Moreove2+, Mn2+, Co2+ or Fe2+ [+/NADH and NADP+/NADPH) and flavin adenine dinucleotide (FAD/FADH2), adenosylcobalamin (AdoCbl), coenzyme A (CoA) or S-adenosyl-l-methionine (SAM), are C5′-substituted adenosine derivatives , so that the functional groups directly involved in the catalyzed reaction could be exposed on the surface of ribozymes ,98,99. OThe increasing complexity of metabolism required more and more information to be encoded in RNA molecules, which, as genetic material, pose two important problems. The first of them is the speed and accuracy of ribozyme-catalyzed replication ,103. TheO-methylation of the d-ribose moiety within an RNA molecule) or potent radical chemistry . The first modification is based on the 2′-O-methylation of the ribose moieties in RNA molecules [O is eradicated via methylation. Consequently, 2′-O-methylated ribonucleotide polymers are stable and resistant to alkaline or enzymatic hydrolysis  of previously synthesized non-modified RNA oligonucleotides. Thus, the modification of all 2′-hydroxyl groups within an RNA molecule would require a high number of transmethylation catalysts (methyltransferase ribozyme or/and enzymes), as they are specific to particular elements of the modified RNA molecule. Moreover, the RNA double helix containing methylated 2′- favored , which m favored ,129. Thu2, H2S H2, N2 and a variety of transition metal cations [2 assimilation and fatty acid synthesis ; N2 assimilation ; numerous redox reactions that are unrelated to gas assimilation  or chemical RNA base modification through SAM-dependent methylation. Interestingly, about 30% of several hundred potential LUCA proteins were S-adenosyl-l-methionine-dependent (SAM-dependent methyltransferases and reductases). The cofactors present in the “RNA-based World” and LUCA are present in all living organisms, regardless of their habitat [The evolution of metabolism from catalytic, self-replicating RNA molecules to simple, single-cell organisms that are capable of information transfer from DNA through RNA to proteins took 200–500 million years. This period was determined based on the analysis of sedimentary rocks containing traces of cellular life fossils, the age of which was estimated to be about 3.5–3.8 billion years ,131,132. cations ,141 that cations ,144,145. cations ,148,149, habitat .N-glycosidic derivatives of β-d-ribofuranose significantly contributed to the formation and development of life on Earth. Furthermore, they are present in the cells of all known organisms as a key element of their metabolism, that is, both catalytic processes and the encoding of genetic information. What contributed to their biological success? Their chemical structure very likely did, from the early stages of metabolic development [d-aldopentose residue serving as a bi-(d-deoxyribose) or trifunctional (d-ribose) conformationally flexible connector on which each nucleotide is formed, and (iii) ionized phosphate residue linking subsequent nucleotides. Some conformational flexibility of the sugar residue enables nucleic acid molecules to take different forms, depending on the nucleotide sequence or environmental conditions, without interfering with the nucleotide base pair geometry. This is particularly noticeable in the case of DNA molecules, for which many different structural forms have been identified [+/NADH, NADP+/NADPH or FAD/FADH2) adenosine-based cofactors. In the “RNA-based World”, nucleic acid molecules have created motifs that enable selective binding of free nucleosides or nucleotides, e.g., adenosine-based cofactors or coribozymes. The shift to enzyme-mediated catalysis did not significantly affect the range of cofactors used, especially adenosine cofactors, which may prove their perfect adaptation to metabolic processes. What changed, though, was only the cofactor and macromolecular interaction type. Unlike coribozymes present on the RNA surface or grooves that are easily accessible from the solvent access region  between a macromolecular environment and coenzyme molecule within the ligand-binding area. The involvement of multiple interactions in the binding cofactors somehow follows their structure, enabling their strong bonding to an active site of an enzyme. It should be noted that during evolution, numerous enzymes that utilize adenosine-based cofactors or substrates have evolutionarily developed the ability to coordinate metal ions, most often in the active site region or in its close proximity. The bond may affect alkaline metal ions , mainly d others ,158,159.d others , S-adenothetases  or S-adedrolases ,162,163.drolases .2, serve as chemical energy storage molecules. Cyclic adenosine monophosphate (cAMP) is a second messenger molecule that is involved in intracellular signal transduction [l-homocysteine  in a cell. The SAM:SAH ratio is perceived as an indicator of the transmethylation activities of the cell [The biological success of ribose-based nucleotides and nucleosides is not limited to encoding genetic information and cellular catalysis nowadays. In addition, these compounds play numerous other roles in metabolic processes that occur in prokaryotic and eukaryotic cells. Some of them are very common, are present in all three domains of life, and are described abundantly in the literature. Briefly, ATP, NADH and FADHsduction . Furthersduction . Biologithe cell  and playthe cell , includithe cell ,169.N-glycosyl derivatives of β-d-ribofuranose, such as SAM, SAH, cAMP and NAD+, are the most abundant [+/NADH and FAD, might be covalently attached to prokaryotic and eukaryotic mRNA to form noncanonical 5′-cap structures [An interesting function of nucleotides and nucleosides is regulating expression that is facilitated through non-covalent or covalent interactions with mRNA molecules. In bacterial cells, a riboswitch, a 5′-UTR region of mRNA molecule, could control the translation process. The riboswitch can bind non-covalently and specifically to appropriate ligands, which induces conformational changes within this 5′-UTR region and, consequently, can modulate transcription termination signals or the availability of ribosome binding sites in these mRNAs ,172,173.abundant ,177,178.ructures . Their bructures ,182,183.The capacity for selective protein–ligand interactions is closely related to the presence of characteristic structural motifs that are responsible for binding individual low-molecular-weight compounds or macromolecules. As it was with the coribozyme-binding aptamers, characteristic motifs/domains selectively binding individual adenine-based nucleotides or nucleosides have evolved as a result of cofactor involvement in enzymatic catalysis . A recenN-ribohydrolyses and transferases utilizing adenosine phosphates are present [S-adenosyl-l-homocysteine (SAH) hydrolysis enzymes that regulate SAM-dependent cellular methylation reactions [S-adenosyl-l-homocysteine (MTA/SAH) nucleosidase, which is a single-domain protein hydrolyzing the MTA (S-methyl-5-thio-d-ribose and adenine or decompose the SAH (S-ribosyl-l-homocysteine. The second enzyme is S-adenosyl-l-homocysteinase (SAHase), which converts SAH to adenosine and homocysteine. It contains two Rossmann folds, one located in the substrate/product (SAH or adenosine)-binding domain and the other in the key cofactor (NAD+)-binding domain  or β-d-deoxyribonucleotide (rarely), which are shown in d-Glu. The enzymes using guanosine-5′-diphosphate (GDP)-based donor molecules involved in the transfer of the mannose (Man) residue from GDP-α-d-Man, the fucose (Fuc) residue from GDP-β-l-Fuc and occasionally glucose (Glu) from GDP-α-d-Glu are slightly more common. However, the vast majority of glycosyltransferases utilize pyrimidine nucleoside phosphate-based donors, predominantly uridine-5′-diphosphate (UDP) derivatives, for transferring numbering sugar residues . The formation of cofactors containing uracil to replace adenine probably occurred at the LUCA stage [d-glucose, dTDP-β-l-rhamnose and dTDP-6-deoxy-4-keto-α-d-glucose). Glycosyltransferases are an extremely diverse group of enzymes, both in terms of sugar residue donors and amino acid sequences that are used. Furthermore, the transfer of the sugar residue can follow two different mechanisms, encompassing either inversion or maintained configuration at the anomeric carbon in the sugar ring. Despite their wide variety, glycosyltransferases utilizing sugar derivatives of nucleoside mono- and diphosphates as donors usually adopttwo structural forms: (i) GT-A containing a single domain  Rossmann fold. Examples of those are glycosyltransferases GT, g,h, a nuCA stage . Glycosye domain g or (ii)e domain h [201,20N-ribohydrolases .” With time, the complexity of the metabolism reaction forced the enzymes to utilize nucleotide cofactors other than adenosine-based derivatives of β-d-ribose.Nucleotides and nucleosides derived from β-


Caenorhabditis elegans (C. elegans) can be effectively explored for students to learn how molecular cues dramatically condition axonal guidance and define nervous system structure and behavior at the organism level. Summary of work: A loosely oriented observational activity preceded detailed discussions on molecules implied in axonal migration. C. elegans mutants were used to introduce second-year medical students to the deleterious effects of gene malfunctioning in neuron response to extracellular biochemical cues and to establish links between molecular function, nervous system structure, and animal behavior. Students observed C. elegans cultures and associated animal behavior alterations with the lack of function of specific axon guidance molecules . Microscopical observations of these strains, in combination with pan-neuronal GFP expression, allowed optimal visualization of severely affected neurons. Once the list of mutated genes in each strain was displayed, students could also relate abnormal patterns in axon migration/ventral and dorsal nerve cord neuron formation in C. elegans with mutated molecular components homologous to those in humans. Summary of results: Students rated the importance and effectiveness of the activity very highly. Ninety-three percent found it helpful to grasp human axonal migration, and all students were surprised with the power of the model in helping to visualize the phenomenon.Aim: Experimental models are a powerful aid in visualizing molecular phenomena. This work reports how the worm  The development of an adequately wired nervous system is essential for the survival of many animal species, including humans. This biological “construction” depends on a tightly regulated genetic program and involves multiple cell proliferation, differentiation, migration, and elimination steps. Wiring of the nervous system also includes extension, specification, and elongation of a particular type of cellular process—the axon—in specific directions, followed by establishing and eliminating synapses, the points of connection between neurons, and a point of significant plasticity even after development. An important component of the proper wiring of nervous systems is the guidance of axonal growth, achieved through the time- and space-specific expression of soluble and diffusible biochemical cues, adhesion molecules, extracellular matrix components, and receptors able to sense those cues and transduce signals to the interior of the axonal process, where cytoskeleton-modifying molecules produce either the advancement of the growth cone (generating an attraction effect) or its collapse (producing a repulsion movement) . A The dysfunction of any of the components of this system generates axonal pathfinding defects that can vary in severity and their functional impact. In more complex nervous systems, there is a significant degree of redundancy. Nevertheless, some genetic mutations affecting the most relevant performers in axon guidance will lead to detectable behavioral phenotypes and nervous system diseases. In humans, these include some autism spectrum disorders and other neurodevelopmental disorders (reviewed in detail in ), and moGiven the complexity of this field of study, it is rarely addressed in undergraduate Biology or Biochemistry programs or during the nervous system courses in medical student training. However, the topic provides a unique opportunity to establish links between nervous system anatomy, biochemistry, and genetics concepts, taking advantage of students’ curiosity towards this very intriguing scientific question. Here, we designed a student-centered activity, combining experimental and virtual components, to promote active learning of key concepts in axonal pathfinding.Caenorhabditis elegans (C. elegans), a nematode for which the structure of the nervous system is very well characterized [C. elegans has 302 neurons categorized into at least 118 different neuronal classes, along with 56 glial cells providing neuronal support [C. elegans encompasses 4887 chemical synapses and 1447 gap junctions [We focused on three prototypic molecules: one soluble cue (netrin/UNC-6) with chemoattractant and chemorepulsive effects on two receptors . We used as a basis for the experimental activity the model organism cterized  , which integrated the contents of human Anatomy, Histology, Physiology, and Biochemistry, in an organic systems-based approach . The actC. elegans netrin ortholog, is expressed by ventral cells  and provides positional information by establishing a ventral-dorsal concentration gradient [unc-6 mutant) are large and healthy but slightly fat and show a kinker motor phenotype. Regarding neuronal wiring, phenotypes include a disorganized ventral nerve cord (VNC), and dorsal extensions of DD and VD neurons that grow in aberrant directions, failing to reach the dorsal nerve cord (DNC) [The activity focused on three prototypical dorsal-ventral axon guidance-related molecules : netrin,gradient ,17,18. Ard (DNC) . C. elegans transmembrane receptors: UNC-40  and UNC-5 . UNC-40 is the major conserved receptor involved in attraction to netrin and binds it directly [unc-40 mutant animals are weak kinker, dumpish, and slow but relatively active [UNC-6 functions through two directly ,20. UNC-y active ,22. unc-5 mutant animals demonstrate comparable growth to wild-type animals but exhibit severe motor defects (uncoordinated phenotype known as severe coiler), DNC is absent or almost absent, cord commissures fail to reach targets, and distribution of cell bodies of the VNC is disorganized. Direct interactions between UNC-40 and UNC-5 cytoplasmic domains are induced by binding to netrin. In the UNC-5/DCC receptor complex, the DCC protein potentiates the UNC-5 repulsive response [Repulsion from UNC-6/netrin primarily depends on the action of the UNC-5 transmembrane receptor, which also binds directly to netrin . UNC-5 iresponse ,24,25.unc-6, unc-5, and unc-40, which exhibit several motor and nervous system defects , including a brief overview of the structure of their nervous system. This was followed by the distribution of Petri dishes with the wild type (WT) and mutant nematode strains (named A to C) and of a score table to fill in  crossed with the quiz strain were projected and printed versions of the photos distributed to students  was provided to the students. The students promptly said that it resembled WT animals. Then, the teacher explained (and showed using a fluorescence stereomicroscope) that the quiz strain expressed a transgene driving the expression of GFP to all students , the aimstudents .Finally, the identity of the mutants was revealed, and the groups (15 min), then the whole class (~20 min) discussed the possible mechanistic relationship between the absence of each specific guidance molecule or receptor and the observed phenotypes, considering their function(s).What is the function of each of the molecules missing in each mutant strain?Name the mammalian orthologue(s) of each molecule.Name mediator(s) of attraction and repulsion signals.unc-5 mutant animals have the DNC almost absent?Why do unc-40 mutant animals have VNC?Why do Prompts for discussion were launched to the students. Some examples are given below:Students were asked to complete the image in -Stereomicroscope ;-Escherichia coli strain OP50 strain;Nematode growth medium (NGM) plates seeded with -Score tables;-Fluorescence stereomicroscope  or pre-acquired images of the strains .C. elegans [Escherichia coli (E. coli) OP50 strain at 20 °C (Standard methods were used for maintaining and culturing  elegans . Nematodat 20 °C .unc-5 (e53), unc-6 (e78), and unc-40 (n324) animals and GFP-positive males were placed on an NGM 30 mm seeded plate at 16 °C for 48 h. Adult hermaphrodites were singled out, and male progeny were evaluated after three days. L4-stage fluorescent animals were singled out, and F2 progeny were selected by UNC phenotypic manifestation and expression of the fluorescent marker . These strains are available upon request.Males of the quiz strain were generated by mating with N2 (WT) males. To generate double mutant animals, hermaphrodites at the L4 stage of mutant unc-6, unc-5, and unc-40 mutant strains were transferred to climatized 60 mm NGM plates seeded with OP50 and allowed to lay eggs for 2 h. After the elimination of the adult animals, eggs were allowed to develop at 20 °C for 4 days. On day 4, plates were renamed mutant A, B, or C and ready for class. For the recording of animals’ motor behavior, the recording system set up comprised a stereomicroscope , a digital camera , and imaging software (Olympus cellSens Entry 3.2 Software). Recordings were performed in 30 mm seeded NGM plates or liquid media (M9 buffer). A 0.8× zoom lens was used to maximize the field of view. The recording was made for 20 s at a rate of 15 frames per second. Recorded images are shown in real-time.Ten gravid adult hermaphrodite animals of WT, unc-6; GFP, unc-5; GFP, unc-40; GFP animals, synchronized by egg laying, were immobilized with 3 mM levamisole  and mounted on a 3% agarose pad. Images of the nervous system of live animals were captured using an Olympus LPS Confocal FV1000 microscope  under a 60× oil (NA = 1.35) objective. Z-series imaging, covering the animal’s total length, was performed using a 488 nm laser excitation line for GFP. The pinhole was adjusted to 1.0 Airy unit of optical slice, and a scan was acquired every ~0.5 μm along the Z-axis. Maximal projection of Z-series images was obtained using Olympus software , and images combined using ImageJ (Fiji v1.5) and Adobe Photoshop  software.Four-day-old n = 44), which happened right after the end of the class. All students answered eight questions about using C. elegans as a model system to understand axonal guidance (4 questions) and the activity (4 questions). Subjects included the second-year medical students who agreed to participate in the rating of the activity (n = 44). The students’ perception of the activity was gathered with eight questions in a Likert 4-points scale , the results of which are shown in C. elegans as a model system to understand axon guidance, nearly all the students agreed that this model allowed the visualization of the biological phenomenon and mimicked the human scenario . The majority of the students also highlighted that there is a good correlation between phenotypes and the genetic mutation underlying it, and considered that the use of C. elegans improved their interest in the course (We used this learning activity as part of a nervous system-focused module over 14 years (2004 to 2018), with very positive informal feedback from participants. We applied a formal activity assessment questionnaire in one of the teaching years (e course A. Regarde course B. This activity has the potential to be used for teaching medical students, as we did here, but also biology, biochemistry, or neuroscience students at the undergraduate or graduate levels. The conversion of this activity into a virtual laboratory could be used on its own for online learning or combined with a laboratory activity to reinforce learning.If taught at the graduate level, the activity can be expanded to additional molecules and molecule types to add diversity and complexity and make each student group have their own project. Concepts, such as the relevance of receptor-receptor interactions, the contribution of adhesion molecules, the role of the extracellular matrix in establishing molecular gradients, and the role of transcriptional regulation and post-translational modifications of the key molecules, can be introduced in these more advanced contexts. This can also be completed using the virtual lab setup if additional components are added.


Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional pre-rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pause-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing. Ribosome biogenesis is influenced by the rate of RNAPI progression. Yet, mechanisms that control RNAPI elongation have remained elusive. Here, the authors show that the conserved protein Seb1 promotes cotranscriptional rRNA processing by controlling RNAPI progression. During the transcription cycle, CTD phosphorylation on Y1, S2, T4, S5, and S7, is tightly controlled by various kinases and phosphatases2, regulating the spatiotemporal recruitment of key factors that control cotranscriptional processing of nascent RNA. For instance, Ser5 phosphorylation on the CTD is essential during early RNA processing events such as capping of nascent mRNAs4, while Ser2 phosphorylation acts in latter stages of cotranscriptional RNA processing, such as during mRNA 3' end maturation and transcription termination7.DNA-directed RNA polymerases are a family of enzymes fundamental for the synthesis of RNA molecules in all living cells, a process known as transcription. In eukaryotic cells, RNA polymerase II (RNAPII) is responsible for the synthesis of messenger (m), small nucleolar (sno), most small nuclear (sn), and a variety of noncoding (nc) RNAs. The production of mature forms of these RNAs requires extensive processing and maturation events, many of which occur as the nascent transcript is being synthesized by RNAPII. The coupling between transcription and RNA processing is largely mediated by the carboxy-terminal domain (CTD) of the RNAPII catalytic subunit, which consists of heptad repeats with the consensus amino acid sequence Y10. As several key steps of RNA maturation occur cotranscriptionally, variations in transcription elongation rates are therefore expected to have important consequences on the efficiency of RNA processing. Indeed, evidence indicate that regulation of RNAPII elongation rates can influence cotranscriptional RNA processing events such as splicing14 and alternative polyadenylation16. One RNAPII-associated factor that appears to influence RNA processing by controlling transcription elongation rate is the fission yeast Seb1 protein. At the amino acid level, S. pombe Seb1 shares homology with S. cerevisiae Nrd1 and human SCAF4/SCAF8 proteins, containing a CTD-interaction domain (CID) as well as a RNA recognition motif (RRM)17. In addition to preferential binding of the Seb1 CID to Ser2-phosphorylated versions of the RNAPII CTD, Seb1 also forms extensive physical contacts with the RNAPII core complex including regions in the vicinity of the RNA exit channel18. These Seb1-polymerase interactions allow cotranscriptional recruitment of Seb1 to RNAPII-transcribed genes, reaching a peak downstream of poly(A) sites where it was shown to associate with nascent pre-mRNAs20. Consistent with binding to nascent pre-mRNAs near the poly(A) signal, depletion of Seb1 causes global 3' UTR lengthening by impairing poly(A) site selection19. Although the exact mechanism by which Seb1 controls poly(A) site selection remains to be determined, reduction of transcription elongation rates attenuates Seb1-dependent 3' UTR length defects, suggesting that Seb1 controls RNA processing by modulating elongation kinetics19. Consistent with this view, NET-seq analysis in fission yeast revealed that Seb1 promotes long-live RNAPII pause states during transcription elongation21.Genome-wide studies indicate that transcription elongation by RNAPII is a rather intermittent process, with frequent occurrences of pausing, backtracking, and arrest23. Yeast RNAPI consist of a 14-subunit complex24 and is the most productive eukaryotic polymerase, contributing up to 60% of the total cellular transcriptional activity25. Transcription by RNAPI is functionally linked to ribosome biogenesis, as RNAPI has only a single genomic target, the multi-copy 9.1 kb-long 35 S rRNA gene. Transcription of the 35 S primary transcript by RNAPI is concurrently associated with pre-rRNA processing, which includes an extensive set of endonucleolytic cleavages and exonucleolytic trimming steps that involve >200 ribosome biogenesis factors26. Specifically, the primary rRNA transcript is processed into a (i) 20 S precursor of the mature 18 S rRNA that is part of the small ribosomal subunit and a (ii) 27 S precursor of the 5.8 S and 25 S rRNAs that make the large ribosomal subunit. Although these endonucleolytic cleavages can occur posttranscriptionally on the released 35 S primary transcript, metabolic labeling assays on short timescales indicate that most pre-rRNA processing occurs cotranscriptionally on nascent pre-rRNAs in yeast27.Besides RNAPII, eukaryotic cells have two additional RNA polymerase complexes: RNAPI and RNAPIII that synthesize most rRNAs and tRNAs, respectively. Based on structural analyses and in vitro assays, all three eukaryotic RNA polymerases share a conserved basic architecture of the RNAP core, whereas peripheral factors are used to promote gene-specific transcription25. Given the substantial amount of cell resources invested in making ribosomes, it is not surprising that ribosome biosynthesis represents a crucial control point in the regulation of cell growth and division29. Regulation of RNAPI transcription is best characterized at the initiation step, where the activity of transcription initiation factors Rrn3 and SL1 is controlled in response to demands in protein synthesis31. In addition, growing evidence support that rRNA synthesis and pre-rRNA processing are also controlled during the elongation phase of RNAPI transcription34. Indeed, recent genomic approaches confirm that transcription elongation at the rDNA is not a continuous process during which RNAPI smoothly traverses the 35 S rRNA gene after initiation, but rather includes frequent RNAPI pausing events and/or regions with slower elongation rates36. Yet, cellular factors and molecular mechanisms that influence the transcription elongation rate of RNAPI remain poorly understood.It is estimated that rapidly dividing yeast cells can generate new ribosomes at a rate of >2000/min, accounting for >75% of the total cellular transcription21. Our findings therefore unveil a role for Seb1 in the control of RNAPI transcription elongation and support a model where Seb1 serves as a general promoter of cotranscriptional RNA processing by decreasing transcription elongation rates.In this study, we show that the conserved fission yeast protein Seb1 associates with the RNAPI transcription machinery and is recruited to the 35 S rRNA gene. Seb1 deficiency results in reduced RNAPI density at the rDNA as well as decreases the cleavage efficiency of the 35 S primary transcript. Whereas the RNA-binding function of Seb1 is essential for its function in the regulation of RNAPI transcription, mutations that impair interaction of Seb1 with phosphorylated versions of the RNAPII CTD did not affect rRNA synthesis and pre-rRNA processing. Importantly, analysis of RNAPI distribution at the single nucleotide resolution revealed that a Seb1 deficiency leads to decreased RNAPI pausing/slow states during transcription elongation, consistent with the role of Seb1 in modulation of RNAPII elongation rate7. In addition to binding to mRNA genes, visual inspection of rDNA revealed Seb1 crosslinking throughout the rRNA gene   Fig. . As posi80) Fig. . The gen19. For the PDB-MS analysis, we generated a strain that expresses Seb1 fused to the TurboID biotin ligase, as we have recently described39. In total, the AP-MS and PDB-MS approaches identified 104819 and 443 Seb1-associated proteins, respectively  and proximity-dependent biotinylation coupled to MS (PDB-MS). The AP-MS analysis of Seb1 was previously described using a fission yeast strain that expressed a HTP (6xHis-TEV-2xProA)-tagged version of Seb1 from its endogenous chromosomal locusely Fig. . In totaely Fig. , includi19 determined by UV-induced RNA-protein cross-linking and analysis of cDNA (CRAC) to assess for direct association of Seb1 with rRNA. By including multi-mapped reads in the analysis to account for the multi-copy nature of rRNA genes, a breakdown of hits from two independent CRAC experiments revealed that nearly 37% of Seb1 binding mapped to rRNA genes  at the rDNA locus in wild-type cells and in a lls Fig. . As a colls Fig. , indicatlls Fig. . To conf42. As expected, ChIP analysis of Rrn3-myc in wild-type cells demonstrated strongest binding at the rDNA promoter  site selection and transcription termination at RNAPII-transcribed genes19. Wild-type and mutant alleles of seb1 were chromosomally integrated as a single copy in the Pnmt1-seb1 conditional strain  and an RNA recognition motif (RRM). We and others have previously used CID and RRM mutants to show that both domains of Seb1 are important for poly(A) site selection and transcription termination at RNAPII-transcribed genesine Fig. . This RRain Fig. , and thetor Fig. . Notablytor Fig. . These r27. These findings could be explained by a faster transcription elongation rate, which is known to modulate cotranscriptional RNA processing43. To test whether Seb1 influences the elongation kinetics of RNAPI in vivo, we determined the distribution of RNAPI at the single nucleotide resolution by CRAC, a high-resolution approach that was previously used to map the elongation profiles of budding and fission yeast RNAPI36. To perform CRAC analysis of RNAPI, we HTP-tagged S. pombe Rpa2 at its endogenous chromosomal locus in both wild-type and Pnmt1-seb1 conditional strains and confirmed the reduction of Rpa2-HTP density at the rDNA in Seb1-deficient cells by ChIP assays . Quantification of relative read counts also revealed substantially decreased pausing over the 5.8 S and ITS2 region  site selection and heterochromatin assembly during RNAPII transcription21, we propose that Seb1 acts as a pause-promoting factor for RNA polymerases I and II to facilitate cotranscriptional RNA processing.Recent analysis of yeast RNAPI distribution at single nucleotide resolution have established that the rate of RNAPI elongation is highly dynamic, varying extensively within the rRNA gene20, we cannot totally exclude the possibility that the effects of the depletion of Seb1 on rRNA processing and RNAPI transcription are indirect. Yet, given that Seb1 physically associated with the RNAPI transcription machinery, was recruited to rDNA , and was shown to crosslink to pre-rRNA, the simplest model is that loss of Seb1 directly influences RNAPI progression, leading to defective cotranscriptional rRNA processing. The fact that Seb1 mutants defective in binding phosphorylated versions of the RNAPII CTD did not impair rRNA processing , our ChIP assays of Rpa2 did not appear to show a biased decrease at the 3' end of the rRNA gene relative to its 5'  and RNAPII21. It is also possible that Seb1 promotes polymerase backtracking, which is a major mechanism underlying pausing of RNA polymerases8. During backtracking, newly transcribed RNAs outside of the polymerase exit channel must re-enter the polymerase catalytic center as the enzyme is slipping backward. The presence of the RNA-binding domain of Seb1 close to the RNA exit channel of the polymerase may prevent the untimely formation of RNA secondary structures, which have been shown to refrain backtracking55. Future studies using in vitro transcription systems should provide insights into the underlying mechanism by which Seb1 promotes long-live RNAP pausing.Seb1 controls the progression of RNAPI (this study) and RNAPII20, our ChIP analysis did not reveal defects in RNAPI termination in Seb1-deficient cells. This was somewhat surprising, as transcription termination of budding yeast RNAPI relies on cotranscriptional RNA cleavage by the dsRNA endoribonuclease Rnt1, which provides entry for the 5'−3' exonuclease Rat1 that degrades RNAPI-associated transcripts and destabilizes the RNAPI transcription complex38. The faster RNAPI progression in Seb1-depleted cells may be expected to impair such cotranscriptional RNA cleavage in the 3'ETS of the rRNA gene, leading to read-through RNAPI. However, we have recently reported that the fission yeast homolog of S. cerevisiae Rnt1, Pac1, is not required for RNAPI termination in S. pombe56, suggesting that fission yeast relies on redundant/alternative mechanisms for termination of RNAPI transcription. One possibility is the “roadblock” mechanism that was previously described in budding yeast58, which involves a DNA-binding factor that promotes transcription termination by pausing/blocking incoming RNAPI at the 3' end of the rDNA. Consistent with this view, our CRAC data revealed piled-up RNAPI at the rDNA 3' end in Seb1-deficicent cells  and RNAPII21, together with the fact that human Seb1 homologs, SCAF4 and SCAF8, also control cotranscriptional mRNA processing by RNAPII60, we predict that the link between transcription elongation kinetics and pre-rRNA processing described in yeast are likely to apply to metazoans.Proliferating yeast and human cells produce around 2000 and 7500 ribosomes per minuteS. pombe strains and oligonucleotides used in this study can be found in Supplementary Table 600nm ~ 0.5–0.8) either in YES (Yeast extract medium) or EMM  supplemented with adenine, histidine, uracil and leucine. For the cells where the seb1 gene was under the thiamine-sensitive nmt1 promoter, treatment with 60 μM of thiamine for 10-14 h were performed to repress transcription. Nuclear depletion of Seb1 was performed using the anchor-away system40 after addition of 2.5 µg/mL rapamycin for 2 h. Mutations in Seb1 were induced by site-directed mutagenesis using primers containing the induced mutations, as previously described19. Addition of C- and N-terminal molecular tags and deletion of genes were performed by a PCR-mediated gene targeting approach61. Expression of proteins with epitope tags were confirmed by western blots and gene deletion was validated by RT-qPCR.A list of 7. Briefly, liquid cultures were grown in EMM to early log phase (OD600nm ∼ 0.3) then rapamycin or an equal volume of DMSO was added to a final concentration of 2.5 μg/ml. After 2 h incubation, nuclei were stained using Hoechst 33342 for 15 min (0.2 mg/ml) and live cells were mounted on 1.2% agarose patches. GFP-tagged proteins and nuclei were detected at 470 nm and 365 nm, respectively, using a Colibri system  on a Zeiss Axio Observer Z1 inverted microscope with a ×100/1.4 oil objective. Data were analyzed using the ZEN black software (Carl Zeiss Canada).Seb1-FRB-GFP localization was detected using fluorescence microscopy as previously described36 from two independent biological replicates. Briefly, strains for CRAC analysis were grown in EMM medium supplemented with thiamine to allow Seb1 depletion. Mid-log exponential phase (OD600nm ~ 0.5–0.8) cells were cross-linked in culture media using megatron UVC cross-linker36 for 100 s. Crosslinked samples were processed and lyzed in TNMC100  with zirconia beads in a 50 mL conical. Cleared lysates were incubated with IgG Sepharose and washed washed three times with TMN600  and two times TMN100 . Protein:RNA complexes were eluted with HaloTEV and the supernatant was subjected to nickel affinity purification in denaturing conditions. After extensive washing of the nickel beads, the addition of 3' linkers, 5' end radiolabeling, and 5' linker ligation was performed on beads. Protein:RNA complexes were then eluted, precipitated, separated on gels, and transferred to nitrocellulose membranes. After detection of labeled RNAs by autoradiography, the appropriate region was excised and treated with proteinase K. The RNA was then isolated, reverse transcribed and PCR amplified as previously described36. Reverse transcription and PCR amplification of wild-type and Seb1-depleted samples were analyzed simultaneously to minimize PCR amplification bias during library preparation. Following gel extraction of PCR amplicons corresponding to 140-200 bp, libraries were measured by Qubit and analyzed by Illumina sequencing.CRAC analysis of Rpa2-HTP was performed as previously described36. Downstream analysis and visualization were performed in Python using pandas, matplotlib and trxtools (https://github.com/TurowskiLab/trxtools). In short, the 3' ends of the reads were counted and pseudocounts were added. The 3' ends of the reads were then transformed into fraction of reads for the transcription unit, smoothed with 30-nt blackman window (pandas) and two biological replicates were averaged. To calculate ratio between datasets, the data were smoothed with 50-nt window, the Seb1-depleted profile was divided by the wild-type profile, and log2 transformation was applied. To calculate the occupancy time . Occupancy time for each position was calculated by multiplication of fraction of reads for each position with The CRAC data was analysed as previously describedTotal cell extracts were resuspended in a cold lysis solution  supplemented with protease inhibitors (1X PMSF and 1X PLAAC). Following the addition of glass beads, lysis on a fast prep  was performed with 3 cycles of 30 s at 6.5 m/s. Final protein concentrations were measured and 30 μg of total proteins were separated on an SDS-PAGE gel, transferred to a nitrocellulose membrane, and analyzed by immunoblotting using either a (i) mouse monoclonal anti-myc  dilution) or anti-Flag  dilution) or a (ii) rabbit polyclonal antibody against protein A  dilution) in the case of HTP-tagged Seb1. Membranes were probes with goat anti-mouse secondary antibodies coupled to AlexaFluor 680  dilution) Protein detection was performed with the Odyssey infrared imaging system at 680 nm (LI-COR).19. For each condition (IP), 50 mL of cell cultures were grown to an OD600nm of ∼0.5–0.8 at 30 °C in EMM + 60 µM of thiamine for 12-15 h. Then, cross-linking was performed by adding a final concentration of 1% formaldehyde in a diluent solution  for 20 min at room temperature (RT) with manual shaking every 5 min. After quenching the solution with glycine , cells were washed twice with a cold Tris-buffer saline (20 mM Tris-HCl [pH 7.5] and 150 mM NaCl) and frozen in liquid nitrogen. Cell pellets were thawed and resuspended in 500 µL of cold lysis buffer  that was supplemented with a protease inhibitor cocktail (1X PMSF and 1X PLAAC) and disrupted vigorously with glass beads in a fast prep  for 3 cycles of 30 s at 6.5 m/s. Then, 150 µL of cold lysis buffer with proteases was added to increase the volume and samples were sonicated for 12 cycles of 10 s at 20% amplitude with a Branson digital sonifier. Whole cell extracts  were then incubated overnight at 4 °C with 50 µl of IgG Dynabeads  in the case of HTP/TAP tagged proteins, or beads coupled with 1.5 ug of mouse monoclonal anti-myc  or 2ug of anti-flag . Then, beads were washed twice with 1 mL of cold lysis buffer, twice with 1 mL of cold lysis buffer with 500 mM NaCl, twice with 1 mL of cold wash buffer  and once with 1 mL of cold Tris-EDTA (TE: 10 mM Tris-HCl [pH 8.0] and 1 mM EDTA [pH8.0]). Bound material was eluted by resuspending the beads in 50 µl elution buffer  at 65 °C for 15 min at 1200 rpm in an Eppendorf Thermomixer. After a short centrifugation, reverse-crosslinking was induced by incubating eluted material with 120 µl of TE and 1% SDS or 5 µl of WCE in 95 µl of TE with 1% SDS at 65 °C overnight. Samples were treated with a proteinase K mix  and DNA was extracted twice with phenol-chloroform-isoamyl alcohol  and once with chloroform (Bioshop), then precipitated with ethanol and resuspended in 30 µl of TE. 10 µg of RNAse A was added to the DNA and after an 1 h incubation at 37 °C, the DNA was purified with a QIAGEN PCR purification kit. The inputs DNA (WCE) was diluted 100 times while the conditions DNA (IP) was diluted 20 times in water. DNA were analyzed on a LightCycler 96 Instrument system (Roche) with the addition of the supermix prerfecta SYBR (QuantaBio) in the presence of 150 mM of gene-specific oligonucleotide. Protein density was calculated by subtracting the mean of three technical replicates of CT conditions (IP) from CT input (WCE). Then, the difference was used to calculate the relative abundance of the immunoprecipitated protein using the 2ΔCT formula.ChIP assays were performed as previously described56. The cells are first resuspended in 500 µl of TES solution  and 500 µl of phenol-chloroform 5:1 (Bioshop) then incubated at 65 °C for 60 min with vortexing every 10 min. The RNA was extracted once with 500 µl of phenol-chloroform 5:1 (Bioshop) and once with 500 µl of chloroform-isoamyl alcohol  then precipitated with ethanol and 3 M NaOAc [pH5.2]. For RT-qPCR analyses, 1 µg of total RNA was treated with DNAse RQ1  and reverse transcribed with RT Omniscript (QIAGEN). The DNA was then diluted 100 times and the expression of the genes was measured on a LightCycler 96 Instrument system (Roche) with the addition of the supermix prerfecta SYBR (QuantaBio) in the presence of 150 mM of gene-specific oligonucleotide  as an internal reference gene. RNA abundance was plotted by normalizing the abundance of the mutant to the abundance of the nda2 gene and either the abundance of a wild-type strain or a specific region.Total RNA was extracted by the acid-phenol heating method, as described previously32P]-ATP using T4 polynucleotide kinase (NEB) for 30 min and heated at 95 °C for 5 min before being added to the membrane. The next day, membranes were washed twice with 2× SSC/0.1% SDS for 5 min and twice with 0.1× SSC/0.1% SDS for 15. A storage phosphor screen  was added to the membrane and photos were taken at different times with a typhoon trio. Quantification was performed with ImageQuantTL software .Following total RNA extraction, 5 μg/well of RNA mixed with a volume of 0.66 of loading buffer were separated on a 0.8% agarose gel containing formaldehyde for 4h30 at 200 V. RNAs were transferred onto a nylon membrane  pre-hybridized in a 5X Denhardt buffer at 42 °C and crosslinked twice. DNA probes were 5' radiolabeled with  and 2% SDS), three times with cold 1X RIPA buffer with addition of 1 mM DTT  and five times with ABC buffer (20 mM ammonium bicarbonate). All washes were done for 5 min at room temperature with agitation. The proteins attached to the beads were then reduced by the addition of 10 mM DTT and alkylated by the addition of 15 mM iodoacetamide (IAA). To digest the proteins still bound to the beads, 1 μg of trypsin was added and incubated at 37 °C overnight and stopped by adding formic acid . Peptides were then extracted twice with acetonitrile, lyophilized and resuspended in 0.1% trifluoroacetic acid (TFA). Desalting was done using ZipTips (EMD Milipore). After a second lyophilization, the peptides were resuspended in 25 µl of TFA then quantified with a nanodrop (Thermo Fisher) at 205 nm.TurboID assay analysis of Seb1 (m/z 350–1600) in profile mode were acquired in the Orbitrap with a resolution of 70,000 after accumulation of 1,000,000 ions. The ten most intense peptide ions from the preview scan in the Orbitrap were fragmented by collision-induced dissociation  after the accumulation of 50,000 ions. Maximal filling times were 250 ms for the full scans and 60 ms for the MS/MS scans. Precursor ion charge state screening was enabled and all unassigned charge states as well as singly, 7 and 8 charged species were rejected. The dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 40 s and a relative mass window of 10 ppm. The lock mass option was enabled for survey scans to improve mass accuracy. Data were acquired using the Xcalibur software version 4.1. Peptide identification was performed with MaxQuant version 1.6.2.2 software using the S. pombe proteome from Uniprot.Trypsin-digested peptides were separated using a Dionex Ultimate 3000 nanoHPLC system. Ten microliters of sample  in 1% (v/v) formic acid were loaded with a constant flow of 4 μl/min onto an Acclaim PepMap100 C18 column . After trap enrichment, peptides were eluted onto an EasySpray PepMap C18 nano column  with a linear gradient of 5–35% solvent B (90% acetonitrile with 0.1% formic acid) over 240 min with a constant flow of 200 nl/min. The HPLC system was coupled to an OrbiTrap QExactive mass spectrometer (Thermo Fisher Scientific Inc) via an EasySpray source. The spray voltage was set to 2.0 kV and the temperature of the column set to 40 °C. Full scan MS survey spectra (Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


In eukaryotic cells, the synthesis, processing, and degradation of mRNA are important processes required for the accurate execution of gene expression programmes. Fully processed cytoplasmic mRNA is characterised by the presence of a 5′cap structure and 3′poly(A) tail. These elements promote translation and prevent non-specific degradation. Degradation via the deadenylation-dependent 5′-3′ degradation pathway can be induced by trans-acting factors binding the mRNA, such as RNA-binding proteins recognising sequence elements and the miRNA-induced repression complex. These factors recruit the core mRNA degradation machinery that carries out the following steps: i) shortening of the poly(A) tail by the Ccr4-Not and Pan2-Pan3 poly (A)-specific nucleases (deadenylases); ii) removal of the 5′cap structure by the Dcp1-Dcp2 decapping complex that is recruited by the Lsm1-7-Pat1 complex; and iii) degradation of the mRNA body by the 5′-3′ exoribonuclease Xrn1. In this review, the biochemical function of the nucleases and accessory proteins involved in deadenylation-dependent mRNA degradation will be reviewed with a particular focus on structural aspects of the proteins and enzymes involved. These fnslation .Cis-acting sequence elements located in the 3′untranslated region (UTR) of mRNAs can induce degradation . The traSaccharomyces cerevisiae, Schizosaccharomyes pombe, Kluyveromyces lactis, Drosophila melanogaster, and Homo sapiens. Here, we will review the structure and function of the core components in deadenylation-dependent 5′-3′ degradation and focus on the wealth of structural insight obtained in the past decade.The components involved in the deadenylation-dependent 5′-3′ degradation pathway are conserved, and detailed insight into the role of the core components has become available through a large number of studies investigating the structure of the enzymes and proteins involved from various model organisms, including 7G cap from the 5′end  mRNA tail using a two-metal catalytic mechanism , removal of this domain does not significantly impair activity . Howeverin vitro . This isin vitro . Howeverin vitro . The Panin vitro . First, in vitro .The Ccr4-Not (carbon catabolite repression–negative on TATA-less) complex is the main deadenylase linked to initiation of mRNA degradation via the 5’-3’ pathway. A number of regulators of mRNA stability have been shown to directly interact with the complex thereby initiating shortening of the poly(A) tail of the mRNA target and initiation of the degradation pathway , as evidenced by purification of CNOT10 and CNOT11 with endogenous Ccr4-Not complexes from human and la cells . Howeverla cells . In the la cells . Evidencla cells . A more la cells   identified two metal sites in the active site, with a preference for Mn2+ and Zn2+ over Mg2+  RNA primarily through backbone interactions  recognition by the two nucleases. Ccr4 selectively recognises poly(A) residues via specific recognition of the adenine bases , whereasractions   or Caf40, is a canonical subunit of the Ccr4-Not complex that acts as a transcriptional co-factor in embryo development, is involved in growth control and cell differentiation, and is associated with tumorigenesis . The DUFCNOT9 is not catalytically active, but structural evidence has shown that it is a hotspot for protein-protein interactions. The interaction of CNOT9 and CNOT1 DUF3819 reveals W-binding pockets on the convex side that can interact with specific tryptophan residues of tristetraprolin (TTP) and GW182/TNRC6 proteins . The armThe ‘NOT module’ of the Ccr4-Not complex located in the C-terminal CTD region of CNOT1 is a trimeric complex with CNOT2 (Not2) and CNOT3 (Not5) . The CTDS. cerevisiae showed that the Ccr4-Not complex is recruited to the ribosome via specific interaction of this Not5 N-terminal domain with the ribosomal E-site, with the requirement that the A-site is empty, tRNA is in the P-site, and the E3 ubiquitin ligase Not4 (CNOT4) is present  is known to form a highly conserved three-helix bundle . Cryo-el present . Binding present .11 shows that each RRM uses a β-sheet bearing highly conserved RNP1 and RNP2 sequence motifs to recognise poly(A) RNA, with the linkers between RRM domains forming a clamp to hold the RNA (The cytoplasmic poly(A)-binding protein 1 (PABPC1) is linked to the Ccr4-Not complex via the BTG/TOB family of proteins. TOB1 and TOB2 interact with the C-terminal MLLE domain of PABPC1 via their PAM2 motifs in their extended C-termini  Figures. BTG1 an the RNA .The accumulation of available structures for the Ccr4-Not nuclease module, BTG2, and PABPC1 with poly(A) RNA have facilitated the construction of a model for the recruitment of Ccr4-Not to mRNA via TOB/BTG and PABPC1 . A combiS. cerevisiae, cells containing loss-of-function alleles of LSm1-7 display impaired mRNA degradation, and oligoadenylated mRNA species accumulate XEEXG (I/L/V)). The disordered C-terminal region contains leucine-rich helical motifs that directly interact with the C-terminal domain of Pat1  for its activity to remove the cap structure and release m7GDP and RNA containing a 5′monophosphate group as the products (7GpppG) or unmethylated cap structures with high affinity , the latter Mg2+ ion may activate a water molecule for nucleophilic attack at the scissile phosphate bond linking the first and second nucleotide . This protein is an RNA helicase involved in miRNA regulation, which also activates the decapping pathway and represses translation . Thus, mIn recent years, a large body of work has established critical steps in the 5′-3′ degradation pathway, including the molecular basis of the catalytic steps required for deadenylation by Pan2-Pan3, decapping, and degradation of the RNA body by Xrn1. In addition, many interactions between the molecular machines involved are understood at the molecular level. Despite immense progress, however, there are still areas that are poorly understood. For example, the requirement for the Caf1 and Ccr4 catalytic subunits during deadenylation by Ccr4-Not, and their collaborative or unique roles are not clear. In addition, the role of uridylation is not understood in detail, and the molecular basis for selective uridylation of oligoadenylated degradation intermediates is not known. A third area for future investigations is to decipher in molecular detail how events in the 5′-3′ mRNA degradation pathway are coordinated with other RNA degradation pathways and the regulation of translational efficiency.


Aspergillus spp. Invasive infections are often life threatening in individuals with weakened immune systems. Detection of Aspergillus galactomannan by enzyme immunoassay (EIA) is the method used most to aid in diagnose aspergillosis. Here, we present the evaluation of a new antigen detection EIA, the MiraVista® Aspergillus antigen detection EIA (MVD EIA) and compare it with the Platelia® Aspergillus EIA.Aspergillosis is a mycosis caused by Retrospective and prospective studies were conducted to evaluate the MVD EIA assay. The retrospective study consisted of bronchoalveolar lavage fluid (BALf) (n=44) from patients diagnosed with aspergillosis in accordance with EROTC guidelines. Controls(n=168) consisted of antigen negative specimens. A prospective study was conducted analyzing the agreement between the Platelia EIA and the MVD EIA. BALf and serum samples were evaluated and a receiver operator curve using Youden’s index was used to determine optimal diagnostic cutoff.The retrospective study using the MVD EIA in BALf demonstrated a sensitivity of 91% in and a specificity of 88%. The prospective study demonstrated a positive agreement between the MVD EIA and the commercial Platelia EIA of 90% and 96% for BAL and serum, respectively. The negative percent agreement was 97% and 96% respectively.The retrospective study demonstrated that the MVD EIA is sensitive and specific when used with BALf. The prospective study demonstrated excellent agreement between the MVD EIA and the commercially available Platelia EIA. The use of this assay has the potential to aid in the diagnosis of aspergillosis.Nicole Bridges, MA, MiraVista Diagnostics: Employee Christopher Dailey, PhD, MiraVista Diagnostics: Employer Andrew Hanzlicek, DVM, MiraVista Diagnostics: employee Joseph Wheat, MD, MiraVista Diagnostics: Ownership Interest|MiraVista Diagnostics: Ownership Interest