Stability of the long noncoding-polyadenylated nuclear (PAN) RNA from Kaposi's sarcoma-associated herpesvirus is conferred by an expression and nuclear retention element (ENE). The ENE protects PAN RNA from a rapid deadenylation-dependent decay pathway via formation of a triple helix between the U-rich internal loop of the ENE and the 3′-poly(A) tail. Because viruses borrow molecular mechanisms from their hosts, we searched highly abundant human long-noncoding RNAs and identified putative ENE-like structures in metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) RNAs. Unlike the PAN ENE, the U-rich internal loops of both predicted cellular ENEs are interrupted by G and C nucleotides and reside upstream of genomically encoded A-rich tracts. We confirmed the ability of MALAT1 and MENβ sequences containing the predicted ENE and A-rich tract to increase the levels of an intronless β-globin reporter RNA. UV thermal denaturation profiles at different pH values support formation of a triple-helical structure composed of multiple U•A-U base triples and a single C•G-C base triple. Additional analyses of the MALAT1 ENE revealed that robust stabilization activity requires an intact triple helix, strong stems at the duplex-triplex junctions, a G-C base pair flanking the triplex to mediate potential A-minor interactions, and the 3′-terminal A of the A-rich tract to form a blunt-ended triplex lacking unpaired nucleotides at the duplextriplex junction. These examples of triple-helical, ENE-like structures in cellular noncoding RNAs, are unique.RNA stability | RNA tertiary interactions

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly-abundant nuclear long noncoding RNA that promotes malignancy. A 3′-stem-loop structure is predicted to confer stability by engaging a downstream A-rich tract in a triple helix, similar to the expression and nuclear retention element (ENE) from the KSHV polyadenylated nuclear RNA. The 3.1-Å resolution crystal structure of the human MALAT1 ENE and A-rich tract reveals a bipartite triple helix containing stacks of five and four U•A-U triples separated by a C+•G-C triplet and C-G doublet, extended by two A-minor interactions. In vivo decay assays indicate that this blunt-ended triple helix, with the 3′ nucleotide in a U•A-U triple, inhibits rapid nuclear RNA decay. Interruption of the triple helix by the C-G doublet induces a “helical reset” that explains why triple-helical stacks longer than six do not occur in nature.

Microtubule-severing enzymes are critical for the biogenesis and maintenance of complex microtubule arrays in axons, spindles, and cilia where tubulin detyrosination, acetylation, and glutamylation are abundant. These modifications exhibit stereotyped patterns suggesting spatial and temporal control of microtubule functions. Using human-engineered and differentially modified microtubules we find that glutamylation is the main regulator of the hereditary spastic paraplegia microtubule severing enzyme spastin. Glutamylation acts as a rheostat and tunes microtubule severing as a function of glutamate number added per tubulin. Unexpectedly, glutamylation is a non-linear biphasic tuner and becomes inhibitory beyond a threshold. Furthermore, the inhibitory effect of localized glutamylation propagates across neighboring microtubules, modulating severing in trans. Our work provides the first quantitative evidence for a graded response to a tubulin posttranslational modification and a biochemical link between tubulin glutamylation and complex architectures of microtubule arrays such as those in neurons where spastin deficiency causes disease.

Acetylation of α-tubulin Lys40 by tubulin acetyltransferase (TAT) is the only known posttranslational modification in the microtubule lumen. It marks stable microtubules and is required for polarity establishment and directional migration. Here we elucidate the mechanistic underpinnings for TAT activity and its preference for microtubules with slow turnover. 1.35 Å TAT cocrystal structures with bisubstrate analogs constrain TAT action to the microtubule lumen and reveal Lys40 engaged in a suboptimal active site. Assays with diverse tubulin polymers show that TAT is stimulated by microtubule inter-protofilament contacts. Unexpectedly, despite the confined intraluminal location of Lys40, TAT efficiently scans the microtubule bidirectionally and acetylates stochastically without preference for ends. First-principles modeling and single-molecule measurements demonstrate that TAT catalytic activity, not constrained luminal diffusion, is rate-limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes.