The discovery of RNA silencing has revealed that non-protein-coding sequences (ncRNAs) can cover essential roles in regulatory networks and their malfunction may result in severe consequences on human health. These findings have prompted a general reassessment of the significance of RNA as a key player in cellular processes. This reassessment, however, will not be complete without a greater understanding of the distribution and function of the over 170 variants of the canonical ribonucleotides, which contribute to the breathtaking structural diversity of natural RNA. This review surveys the analytical approaches employed for the identification, characterization, and detection of RNA posttranscriptional modifications (rPTMs). The merits of analyzing individual units after exhaustive hydrolysis of the initial biopolymer are outlined together with those of identifying their position in the sequence of parent strands. Approaches based on next generation sequencing and mass spectrometry technologies are covered in depth to provide a comprehensive view of their respective merits. Deciphering the epitranscriptomic code will require not only mapping the location of rPTMs in the various classes of RNAs, but also assessing the variations of expression levels under different experimental conditions. The fact that no individual platform is currently capable of meeting all such demands implies that it will be essential to capitalize on complementary approaches to obtain the desired information. For this reason, the review strived to cover the broadest possible range of techniques to provide readers with the fundamental elements necessary to make informed choices and design the most effective possible strategy to accomplish the task at hand.

Human J-domain protein (JDP) DnaJB6 has a broad and potent activity that prevents formation of amyloid by polypeptides such as polyglutamine, A-beta, and alpha-synuclein, related to Huntington's, Alzheimer's, and Parkinson's diseases, respectively. In yeast, amyloid-based [PSI+] prions, which rely on the related JDP Sis1 for replication, have a latent toxicity that is exposed by reducing Sis1 function. Anti-amyloid activity of DnaJB6 is very effective against weak [PSI+] prions and the Sup35 amyloid that composes them, but ineffective against strong [PSI+] prions composed of structurally different amyloid of the same Sup35. This difference reveals limitations of DnaJB6 that have implications regarding its therapeutic use for amyloid disease. Here, we find that when Sis1 function is reduced, DnaJB6 represses toxicity of strong [PSI+] prions and inhibits their propagation. Both Sis1 and DnaJB6, which are regulators of protein chaperone Hsp70, counteract the toxicity by reducing excessive incorporation of the essential Sup35 into prion aggregates. However, while Sis1 apparently requires interaction with Hsp70 to detoxify [PSI+], DnaJB6 counteracts prion toxicity by a different, Hsp70-independent mechanism.

The accumulation of misfolded proteins as amyloids is associated with pathology in dozens of debilitating human disorders, including diabetes, Alzheimer’s, Parkinson’s, and Huntington’s diseases. Expressing human amyloid-forming proteins in yeast is toxic, and yeast prions that propagate as infectious amyloid forms of cellular proteins are also harmful. The yeast system, which has been useful for studying amyloids and their toxic effects, has provided much insight into how amyloids affect cells and how cells respond to them. Given that an amyloid is a protein folding problem, it is unsurprising that the factors found to counteract the propagation or toxicity of amyloids in yeast involve protein quality control. Here, we discuss such factors with an emphasis on J-domain proteins (JDPs), which are the most highly abundant and diverse regulators of Hsp70 chaperones. The anti-amyloid effects of JDPs can be direct or require interaction with Hsp70.

[PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI+] prions.

18 [PSI + ] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In 19 [PSI + ] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, 20 [PSI + ] prions typically affect viability only modestly, so [PSI + ] must balance sequestering Sup35 21 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J22 protein regulator of Hsp70 required for propagation of amyloid-based yeast prions. C-terminally 23 truncated Sis1 (Sis1JGF) supports cell growth in place of wild type Sis1. Sis1JGF also supports 24 [PSI + ] propagation, yet [PSI + ] is highly toxic to cells expressing only Sis1JGF. We searched 25 extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is 26 uniquely needed to protect from [PSI + ] toxicity. We find the C-terminal substrate-binding 27 domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI + ] cells 28 that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion 29 aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions 30 relieved [PSI + ] toxicity. Together our data suggest that Sis1 has separable roles in propagating 31 Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for 32 propagation of what otherwise would be lethal [PSI + ] prions. 33 D ow naded rom http/academ ic.p.com /genetics/advance-a.1093/genetics/iyab129/6345347 by gest on 12 Agust 2021 10692_0_art_file_251274_qxbnjp.docx 3 Introduction 34 Prions are infectious misfolded proteins associated with pathology in many organisms 35 (MCGLINCHEY et al. 2011; CHERNOVA et al. 2017; CHITI AND DOBSON 2017; SOTO AND 36 PRITZKOW 2018). The [PSI + ] prion of S. cerevisiae propagates as insoluble amyloid of the 37 essential ribosome release factor Sup35 (STANSFIELD et al. 1995; ZHOURAVLEVA et al. 1995; 38 TUITE AND COX 2007; WICKNER 2016). Sup35 contains a dispensable N-terminal region, 39 separable from the functional domain, that forms the amyloid core of prion fibers (TER40 AVANESYAN et al. 1994; KING et al. 1997; PAUSHKIN et al. 1997a). This prion-determining 41 region of Sup35 is followed by a dispensable charged middle region and the C-terminal domain 42 (CTD) that functions in translation termination (TER-AVANESYAN et al. 1994; LIEBMAN AND 43 CHERNOFF 2012). Cells propagating [PSI + ] display a nonsense suppressor phenotype caused by 44 reduced ability of Sup35 to promote release of ribosomes at termination codons of mRNAs. This 45 obvious phenotype makes yeast an ideal system for monitoring cellular processes that promote or 46 inhibit ability of prions to propagate in cells. 47 Sup35, like other amyloid-forming proteins, can form amyloids with different physical 48 characteristics (TOYAMA et al. 2007). Variants of [PSI + ] prions display different strength and 49 mitotic stability phenotypes that reflect such differences in the underlying Sup35 amyloids 50 (DERKATCH et al. 1996; TANAKA et al. 2006). A stronger nonsense suppressor phenotype reflects 51 a greater reduction in efficiency of translation termination, which correlates with the extent 52 Sup35 is depleted into prion aggregates (ZHOU et al. 1999; JUNG et al. 2000; UPTAIN et al. 53 2001). In turn, the degree of aggregation of Sup35 depends on the rates of assembly and division 54 of Sup35 amyloid (TANAKA et al. 2004; TANAKA et al. 2006). The division rate determines the 55 number of prion "seeds" per cell, which often correlates with strength of phenotype as it 56 D ow naded rom http/academ ic.p.com /genetics/advance-a.1093/genetics/iyab129/6345347 by gest on 12 Agust 2021 10692_0_art_file_251274_qxbnjp.docx 4 determines the number of amyloid ends available to recruit Sup35 into insoluble prion polymers. 57 This seed number also can determine the efficiency by which the seeds are transmitted as cells 58 divide, and therefore the mitotic stability of a prion. Replication of prions by division depends on 59 the same Hsp104-driven machinery that re-solubilizes proteins from aggregates to help cells 60 recover from protein-denaturing stresses (CHERNOFF et al. 1995; PAUSHKIN et al. 1996; GLOVER 61 AND LINDQUIST 1998; LUM et al. 2004; HUNG AND MASISON 2006; TIPTON et al. 2008; REIDY et 62 al. 2012). Hsp104 function requires the Hsp70 system and various components of this machinery 63 influence prion propagation and elimination (MASISON AND REIDY 2015; CHERNOVA et al. 64 2017). 65 Hsp70 is a key player in protein quality control that acts in protein translation, folding, 66 degradation and translocation across membranes (SHARMA AND MASISON 2009; CRAIG 2018; 67 ROSENZWEIG et al. 2019). Hsp70 function relies on J-proteins that recruit substrates to Hsp70 68 and stimulate its ATP hydrolysis (FAN et al. 2003). Altering J-proteins and many other Hsp70 69 co-chaperones that prions depend on can perturb prion propagation, possibly through effects on 70 Hsp104 activity (MORIYAMA et al. 2000; SONDHEIMER et al. 2001; KRYNDUSHKIN et al. 2002; 71 JONES et al. 2004; KRYNDUSHKIN AND WICKNER 2007; REIDY AND MASISON 2011; REIDY et al. 72 2012). Among J-proteins, only Sis1 is essential for both cell viability and for propagation of the 73 most widely studied yeast prions (LUKE et al. 1991; HIGURASHI et al. 2008). It possesses an N74 terminal J-domain that binds Hsp70 and activates Hsp70 ATPase, adjacent glycine75 phenylalanine (GF) and glycine-methionine (GM) rich regions that contribute to functional 76 specificity, and a C-terminal substrate-binding domain (CTD) that ends in a dimerization domain 77 (DD) (YAN AND CRAIG 1999; LI et al. 2009). Although some prions are exquisitely sensitive to 78 alterations in Sis1 function (REIDY et al. 2014), propagation of [PSI + ] and [PIN + ] is supported by 79 D ow naded rom http/academ ic.p.com /genetics/advance-a.1093/genetics/iyab129/6345347 by gest on 12 Agust 2021 10692_0_art_file_251274_qxbnjp.docx 5 Sis1 lacking all but its J and GF regions (LOPEZ et al. 2003; KIRKLAND et al. 2011; HARRIS et al. 8

Canonically, heterochromatin formation in fission yeast and metazoans is initiated by di/trimethylation of histone H3 at lysine 9 position by the histone methyltransferase Suv39H1/Clr4, followed by binding of Swi6/HP1 to H3-K9-me2/me3 via its chromodomain. Subsequent self-association of Swi6/HP1 on adjacent nucleosomes or a cooperative interaction between Clr4 and Swi6/HP1 leads to folded heterochromatin structure. HP1 binding to RNA is shown to facilitate its localization at and assembly of heterochromatin in metazoans. Likewise, recruitment of Swi6/HP1 to centromere depends on the RNAi pathway in fission yeast; paradoxically, Swi6/HP1 is also thought to play a role in RNA turnover. Here we provide evidence in support of RNAi-independent recruitment of Swi6. We show that, apart from the low affinity binding to RNAs through its hinge domain, as already reported, Swi6/HP1 displays a hierarchy of increasing binding affinity through its chromodomain to the siRNAs corresponding to specific dg-dh repeats and even stronger binding to the cognate siRNA-DNA hybrids than to the siRNA precursors or general RNAs. Our results support a mechanism of recruitment of Swi6, which is dependent on its specific and high affinity binding to siRNA-DNA hybrid at the dg-dh repeats. This binding, which is independent of, albeit augmented by binding to H3-K9-Me2, leads to heterochromatin formation and silencing. We suggest that the net role of Swi6 in RNA physiology may be regulated by a balance between abundance and affinity of Swi6 towards heterochromatic and euchromatic RNAs and siRNAs.

Polyglutamine (polyQ) aggregates are associated with pathology in protein-folding diseases and with toxicity in the yeast Saccharomyces cerevisiae Protection from polyQ toxicity in yeast by human DnaJB6 coincides with sequestration of aggregates. Gathering of misfolded proteins into deposition sites by protein quality control (PQC) factors has led to the view that PQC processes protect cells by spatially segregating toxic aggregates. Whether DnaJB6 depends on this machinery to sequester polyQ aggregates, if this sequestration is needed for DnaJB6 to protect cells, and the identity of the deposition site are unknown. Here, we found DnaJB6-driven deposits share characteristics with perivacuolar insoluble protein deposition sites (IPODs). Binding of DnaJB6 to aggregates was necessary, but not enough, for detoxification. Focal formation required a DnaJB6-Hsp70 interaction and actin, polyQ could be detoxified without sequestration, and segregation of aggregates alone was not protective. Our findings suggest DnaJB6 binds to smaller polyQ aggregates to block their toxicity. Assembly and segregation of detoxified aggregates are driven by an Hsp70- and actin-dependent process. Our findings show sequestration of aggregates is not the primary mechanism by which DnaJB6 suppresses toxicity and raise questions regarding how and when misfolded proteins are detoxified during spatial segregation.