Dimeric assembly of human Suv3 helicase promotes its RNA unwinding function in mitochondrial RNA degradosome for RNA decay.

Human Suv3 is a unique homodimeric helicase that constitutes the major component of the mitochondrial degradosome to work cooperatively with exoribonuclease PNPase for efficient RNA decay. However, the molecular mechanism of how Suv3 is assembled into a homodimer to unwind RNA remains elusive. Here, we show that dimeric Suv3 preferentially binds to and unwinds DNA-DNA, DNA-RNA, and RNA-RNA duplexes with a long 3' overhang (≥10 nucleotides). The C-terminal tail (CTT)-truncated Suv3 (Suv3ΔC) becomes a monomeric protein that binds to and unwinds duplex substrates with ~six to sevenfold lower activities relative to dimeric Suv3. Only dimeric Suv3, but not monomeric Suv3ΔC, binds RNA independently of ATP or ADP, and is capable of interacting with PNPase, indicating that dimeric Suv3 assembly ensures its continuous association with RNA and PNPase during ATP hydrolysis cycles for efficient RNA degradation. We further determined the crystal structure of the apo-form of Suv3ΔC, and SAXS structures of dimeric Suv3 and PNPase-Suv3 complex, showing that dimeric Suv3 caps on the top of PNPase via interactions with S1 domains, and forms a dumbbell-shaped degradosome complex with PNPase. Overall, this study reveals that Suv3 is assembled into a dimeric helicase by its CTT for efficient and persistent RNA binding and unwinding to facilitate interactions with PNPase, promote RNA degradation, and maintain mitochondrial genome integrity and homeostasis.

Frontotemporal dementia-linked P112H mutation of TDP-43 induces protein structural change and impairs its RNA binding function.

TDP-43 forms the primary constituents of the cytoplasmic inclusions contributing to various neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Over 60 TDP-43 mutations have been identified in patients suffering from these two diseases, but most variations are located in the protein's disordered C-terminal glycine-rich region. P112H mutation of TDP-43 has been uniquely linked to FTD, and is located in the first RNA recognition motif (RRM1). This mutation is thought to be pathogenic, but its impact on TDP-43 at the protein level remains unclear. Here, we compare the biochemical and biophysical properties of TDP-43 truncated proteins with or without P112H mutation. We show that P112H-mutated TDP-43 proteins exhibit higher thermal stability, impaired RNA-binding activity, and a reduced tendency to aggregate relative to wild-type proteins. Near-UV CD, 2D-nuclear-magnetic resonance, and intrinsic fluorescence spectrometry further reveal that the P112H mutation in RRM1 generates local conformational changes surrounding the mutational site that disrupt the stacking interactions of the W113 side chain with nucleic acids. Together, these results support the notion that P112H mutation of TDP-43 contributes to FTD through functional impairment of RNA metabolism and/or structural changes that curtail protein clearance.

RNA recognition motifs of disease-linked RNA-binding proteins contribute to amyloid formation.

Aberrant expression, dysfunction and particularly aggregation of a group of RNA-binding proteins, including TDP-43, FUS and RBM45, are associated with neurological disorders. These three disease-linked RNA-binding proteins all contain at least one RNA recognition motif (RRM). However, it is not clear if these RRMs contribute to their aggregation-prone character. Here, we compare the biophysical and fibril formation properties of five RRMs from disease-linked RNA-binding proteins and five RRMs from non-disease-associated proteins to determine if disease-linked RRMs share specific features making them prone to self-assembly. We found that most of the disease-linked RRMs exhibit reversible thermal unfolding and refolding, and have a slightly lower average thermal melting point compared to that of normal RRMs. The full domain of TDP-43 RRM1 and FUS RRM, as well as the ß-peptides from these two RRMs, could self-assemble into fibril-like aggregates which are amyloids of parallel ß-sheets as verified by X-ray diffraction and FT-IR spectroscopy. Our results suggest that some disease-linked RRMs indeed play important roles in amyloid formation and shed light on why RNA-binding proteins with RRMs are frequently identified in the cellular inclusions of neurodegenerative diseases.

Structural insights into nanoRNA degradation by human Rexo2.

Human RNA exoribonuclease 2 (Rexo2) is an evolutionarily conserved 3'-to-5' DEDDh-family exonuclease located primarily in mitochondria. Rexo2 degrades small RNA oligonucleotides of <5 nucleotides (nanoRNA) in a way similar to Escherichia coli Oligoribonuclease (ORN), suggesting that it plays a role in RNA turnover in mitochondria. However, how Rexo2 preferentially binds and degrades nanoRNA remains elusive. Here, we show that Rexo2 binds small RNA and DNA oligonucleotides with the highest affinity, and it is most robust in degrading small nanoRNA into mononucleotides in the presence of magnesium ions. We further determined three crystal structures of Rexo2 in complex with single-stranded RNA or DNA at resolutions of 1.8-2.2 Å. Rexo2 forms a homodimer and interacts mainly with the last two 3'-end nucleobases of substrates by hydrophobic and π-π stacking interactions via Leu53, Trp96, and Tyr164, signifying its preference in binding and degrading short oligonucleotides without sequence specificity. Crystal structure of Rexo2 is highly similar to that of the RNA-degrading enzyme ORN, revealing a two-magnesium-ion-dependent hydrolysis mechanism. This study thus provides the molecular basis for human Rexo2, showing how it binds and degrades nanoRNA into nucleoside monophosphates and plays a crucial role in RNA salvage pathways in mammalian mitochondria.

Crystal structure of dimeric human PNPase reveals why disease-linked mutants suffer from low RNA import and degradation activities.

Human polynucleotide phosphorylase (PNPase) is an evolutionarily conserved 3'-to-5' exoribonuclease principally located in mitochondria where it is responsible for RNA turnover and import. Mutations in PNPase impair structured RNA transport into mitochondria, resulting in mitochondrial dysfunction and disease. PNPase is a trimeric protein with a doughnut-shaped structure hosting a central channel for single-stranded RNA binding and degradation. Here, we show that the disease-linked human PNPase mutants, Q387R and E475G, form dimers, not trimers, and have significantly lower RNA binding and degradation activities compared to wild-type trimeric PNPase. Moreover, S1 domain-truncated PNPase binds single-stranded RNA but not the stem-loop signature motif of imported structured RNA, suggesting that the S1 domain is responsible for binding structured RNAs. We further determined the crystal structure of dimeric PNPase at a resolution of 2.8 Å and, combined with small-angle X-ray scattering, show that the RNA-binding K homology and S1 domains are relatively inaccessible in the dimeric assembly. Taken together, these results show that mutations at the interface of the trimeric PNPase tend to produce a dimeric protein with destructive RNA-binding surfaces, thus impairing both of its RNA import and degradation activities and leading to mitochondria disorders.

Structural insights into RNA unwinding and degradation by RNase R.

RNase R is a conserved exoribonuclease in the RNase II family that primarily participates in RNA decay in all kingdoms of life. RNase R degrades duplex RNA with a 3' overhang, suggesting that it has RNA unwinding activity in addition to its 3'-to-5' exoribonuclease activity. However, how RNase R coordinates RNA binding with unwinding to degrade RNA remains elusive. Here, we report the crystal structure of a truncated form of Escherichia coli RNase R (residues 87-725) at a resolution of 1.85 Å. Structural comparisons with other RNase II family proteins reveal two open RNA-binding channels in RNase R and suggest a tri-helix 'wedge' region in the RNB domain that may induce RNA unwinding. We constructed two tri-helix wedge mutants and they indeed lost their RNA unwinding but not RNA binding or degrading activities. Our results suggest that the duplex RNA with an overhang is bound in the two RNA-binding channels in RNase R. The 3' overhang is threaded into the active site and the duplex RNA is unwound upon reaching the wedge region during RNA degradation. Thus, RNase R is a proficient enzyme, capable of concurrently binding, unwinding and degrading structured RNA in a highly processive manner during RNA decay.

Mycobacterium avium subsp. paratuberculosis is not discerned in diabetes mellitus patients in Hyderabad, India.

Mycobacterium avium ssp. paratuberculosis (MAP) is an obligate intracellular pathogen. It causes chronic intestinal inflammation in ruminants known as Johne's disease and is associated with human Crohn's disease. Furthermore, association of MAP with other autoimmune diseases, such as type-1 diabetes, has been established in patients from Sardinia (Italy) which is a MAP endemic and genetically isolated region. Due to largest livestock population and consequently high MAP prevalence amidst a very high diabetes incidence in India, we sought to test this association on a limited number of patient samples from Hyderabad. Our results of ELISA with MAP lysate and MAP-specific protein MAP3738c as well as PCR/real-time PCR of MAP-specific sequences IS900 and/or f57 indicated that, in contrast to Sardinian diabetic patients, MAP infection in blood is not discerned in diabetic patients in Hyderabad. The association of a mycobacterial trigger with diabetes therefore could well be a population-specific phenomenon, highly dependent on genetic repertoire and the environment of susceptible populations. However, a larger study is needed in order to confirm this.