Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3′→5′ exonuclease containing S1 and KH RNA-binding domains

The exosome, an evolutionarily conserved complex of multiple 3′→5′ exoribonucleases, is responsible for a variety of RNA processing and degradation events in eukaryotes. In this report Arabidopsis thaliana AtRrp4p is shown to be an active 3′→5′ exonuclease that requires a free 3′-hydroxyl and degrades RNA hydrolytically and distributively, releasing nucleoside 5′-monophosphate products. AtRrp4p behaves as an ~500 kDa species during sedimentation through a 10–30% glycerol gradient, co-migrating with AtRrp41p, another exosome subunit, and it interacts in vitro with AtRrp41p, suggesting that it is also present in the plant cell as a subunit of the exosome. We found that, in addition to a previously reported S1-type RNA-binding domain, members of the Rrp4p family of proteins contain a KH-type RNA-binding domain in the C-terminal half and show that either domain alone can bind RNA. However, only the full-length protein is capable of degrading RNA and interacting with AtRrp41p.     

Mitogen-activated protein kinase p38 and MK2, MK3 and MK5: Ménage à trois or ménage à quatre?

The mitogen-activated protein kinase (MAPK) signalling pathways play pivotal roles in cellular processes such as proliferation, apoptosis, gene regulation, differentiation, and cell motility. The typical mammalian MAPK pathways ERK1/2, JNK, p38^MAPK, and ERK5 operate through a concatenation of three successive phosphorylation events mediated by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs phosphorylate substrates with distinct functions, including other protein kinases referred to as MAPK-activated protein kinases. One family of related MAPK-activated protein kinases includes MK2, MK3, and MK5. While it is generally accepted that MK2 and MK3 are bona fide substrates for p38^MAPK, the genuineness of MK5 as a p38^MAPK substrate is disputed. This review summarizes the findings pro and contra an authentic p38^MAPK-MK5 relationship, discusses possible explanations for these discrepancies, and proposes experiments that may help to unequivocally clarify whether MK5 is indeed a substrate for p38^MAPK.     

Distinct roles of MK2 and MK5 in cAMP/PKA- and stress/p38MAPK-induced heat shock protein 27 phosphorylation

Background

Classical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three successive phosphorylation events resulting in the phosphorylation of a variety of substrates, including another class of protein kinases referred to as MAPK-activating protein kinases (MAPKAPKs). The MAPKAPKs MK2, MK3 and MK5 are closely related, but MK2 and MK3 are the major downstream targets of the p38^MAPK pathway, while MK5 can be activated by the atypical MAPK ERK3 and ERK4, protein kinase A (PKA), and maybe p38^MAPK. MK2, MK3, and MK5 can phosphorylate the common substrate small heat shock protein 27 (HSP27), a modification that regulates the role of HSP27 in actin polymerization. Both stress and cAMP elevating stimuli can cause F-actin remodeling, but whereas the in vivo role of p38^MAPK-MK2 in stress-triggered HSP27 phosphorylation and actin reorganization is well established, it is not known whether MK2 is involved in cAMP/PKA-induced F-actin rearrangements. On the other hand, MK5 can phosphorylate HSP27 and cause cytoskeletal changes in a cAMP/PKA-dependent manner, but its role as HSP27 kinase in stress-induced F-actin remodeling is disputed. Therefore, we wanted to investigate the implication of MK2 and MK5 in stress- and PKA-induced HSP27 phosphorylation.

Results

Using HEK293 cells, we show that MK2, MK3, and MK5 are expressed in these cells, but MK3 protein levels are very moderate. Stress- and cAMP-elevating stimuli, as well as ectopic expression of active MKK6 plus p38^MAPK or the catalytic subunit of PKA trigger HSP27 phosphorylation, and specific inhibitors of p38^MAPK and PKA prevent this phosphorylation. Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels. Stimulation of the p38^MAPK, but not the PKA pathway, caused activation of MK2.

Conclusion

Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway. Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization.     

Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5′-phosphate and 3′-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3′-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3′p substrate to generate an RNA 2′,3′-cyclic phosphate or convert DNA3′p to ssDNA^3′pp^5′A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3′p. These modifications of RNA and DNA 3′-phosphates are similar to the activities of RtcA, an RNA 3′-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3′p or DNA 3′p to generate the adenylated intermediate. For RNA ^3′pp^5′A, the third step involves attack of the adjacent 2′ hydroxyl to generate the RNA 2′,3′-cyclic phosphate. These steps are analogous to those in classical 5′ phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3′p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3′-phosphorylated nicks in double-stranded DNA to produce a 3′-adenylated product. These 3′-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5′Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3′-terminal phosphates.     

p38/p53-Mediated Bax induction contributes to neurons degeneration in rotenone-induced cellular and rat models of Parkinson’s disease

Rotenone is an environmental neurotoxin that induces degeneration of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc), which ultimately results in parkinsonism, but the molecular mechanisms of selective degeneration of nigral DA neurons are not fully understood. In the present study, we investigated the induction of p38^MAPK/p53 and Bax in SNpc of Lewis rats after chronic treatment with rotenone and the contribution of Bax to rotenone-induced apoptotic commitment of differentiated PC12 cells. Lewis rats were subcutaneously treated with rotenone (1.5 mg/kg) twice a day for 50 days and the loss of tyrosine hydroxylase (THase), motor function impairment, and expression of p38^MAPK, P-p38^MAPK, p53, and Bax were assessed. After differentiated PC cells were treated with rotenone (500 nM) for 6–36 h, protein levels of p38^MAPK and P-p38^MAPK, p53 nuclear translocation, Bax induction and cell death were measured. The results showed that rotenone administration significantly reduced motor activity and caused a loss of THase immunoreactivity in SNpc of Lewis rats. The degeneration of nigral DA neurons was accompanied by the increases in p38^MAPK, P-p38^MAPK, p53, and Bax protein levels. In cultured PC12 cells, rotenone also induced an upregulation of p38^MAPK, P-p38^MAPK, p53 and Bax. Pharmacological inhibition of p38^MAPK with SB203580 (25 μM) blunted rotenone-induced cell apoptosis. Treatment with SB203580 prevented the p53 nuclear translocation and upregulation of Bax. Inhibition of p53 with pifthrin-alpha or Bax with siRNAs significantly reduced rotenone-induced Bax induction and apoptotic cell death. These results suggest that the p38^MAPK/p53-dependent induction of Bax contributes to rotenone’s neurotoxicity in PD models.     

MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation

The exosome is a complex of 3′→5′ exoribonucleases which is involved in many RNA metabolic processes. To regulate these functions distinct proteins are believed to recruit the exosome to specific substrate RNAs. Here, we demonstrate that M-phase phosphoprotein 6 (MPP6), a protein reported previously to co-purify with the TAP-tagged human exosome, accumulates in the nucleoli of HEp-2 cells and associates with a subset of nuclear exosomes as evidenced by co-immunoprecipitation and biochemical fractionation experiments. In agreement with its nucleolar accumulation, siRNA-mediated knock-down experiments revealed that MPP6 is involved in the generation of the 3′ end of the 5.8S rRNA. The accumulation of the same processing intermediates after reducing the levels of either MPP6 or exosome components strongly suggests that MPP6 is required for the recruitment of the exosome to the pre-rRNA. Interestingly, MPP6 appeared to display RNA-binding activity in vitro with a preference for pyrimidine-rich sequences, and to bind to the ITS2 element of pre-rRNAs. Our data indicate that MPP6 is a nucleolus-specific exosome co-factor required for its role in the maturation of 5.8S rRNA.     

Trypanosome MTR4 is involved in rRNA processing

The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the ‘TRAMP’ complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p. Depletion of the Trypanosoma brucei MTR4-like protein TbMTR4 caused growth arrest and defects in 5.8S rRNA processing similar to those seen after depletion of the exosome. TbNPAPL, a nuclear protein which is a putative homolog of Trf4p/Pap2p, was required for normal cell growth. Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect. These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution. In contrast, there was no evidence for a trypanosome TRAMP complex since no stable interactions between TbMTR4 and the exosome, TbNPAPL or RNA-binding proteins were detected.     

A redox microenvironment is essential for MAPK-dependent secretion of pro-inflammatory cytokines: modulation by glutathione (GSH/GSSG) biosynthesis and equilibrium in the alveolar epithelium

The characterization of oxidant (glutathione)-dependent regulation of MAPK^p38/RK-mediated TNF-α secretion was undertaken in vitro, and the ramifications of the influence of a redox microenvironment were unraveled. Intermittent exposure of alveolar epithelial cells (FATEII) to LPS (endotoxin) transiently and temporally induced the expression of MAPK^p38/RK. This upregulation was associated with the activation of MAPKAP-K₂, manifested by the specific phosphorylation of the downstream heat-shock protein (Hsp)-27. Selective blockading of the MAPK^p38/RK pathway using the pyridinyl imidazole SB-203580 abrogated the LPS-dependent release of TNF-α. N-acetyl-l-cysteine (NAC), a precursor of glutathione, reduced TNF-α secretion and increased [GSH]. Conversely, l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in the pathway mediating GSH biosynthesis, augmented the secretion of TNF-α and [GSSG] accumulation. Whereas NAC abrogated the phosphorylation of MAPK^p38/RK, BSO reversibly amplified this effect. Furthermore, intermittent exposure of FATEII cells to the exogenous oxidants X/XO and H₂O₂ upregulated the secretion of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α; this upregulation was correlated with increasing activity of key glutathione-related enzymes, closely involved with maintaining the cyclic GSH/GSSG equilibrium. These results indicate that a redox microenvironment plays a major role in regulating MAPK-dependent production of cytokines in the alveolar epithelium.     

2′-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa

RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3′-phosphate RNA end to a 2′,3′-cyclic phosphate via covalent enzyme-(histidinyl-Nϵ)-AMP and RNA(3′)pp(5′)A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2′-phosphate RNA end in high yield. The rate of 2′-phosphate cyclization by RtcA is five orders of magnitude slower than 3′-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA_3′p and RNA_2′p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5′-OH ends to either 3′-phosphate or 2′,3′-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2′-phosphates, which cannot be spliced by RtcB, to 2′,3′-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ⁵⁴ coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon.     

WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N7-methylation of G1639 in human 18S rRNA

Ribosomal (r)RNAs are extensively modified during ribosome synthesis and their modification is required for the fidelity and efficiency of translation. Besides numerous small nucleolar RNA-guided 2′-O methylations and pseudouridinylations, a number of individual RNA methyltransferases are involved in rRNA modification. WBSCR22/Merm1, which is affected in Williams–Beuren syndrome and has been implicated in tumorigenesis and metastasis formation, was recently shown to be involved in ribosome synthesis, but its molecular functions have remained elusive. Here we show that depletion of WBSCR22 leads to nuclear accumulation of 3′-extended 18SE pre-rRNA intermediates resulting in impaired 18S rRNA maturation. We map the 3′ ends of the 18SE pre-rRNA intermediates accumulating after depletion of WBSCR22 and in control cells using 3′-RACE and deep sequencing. Furthermore, we demonstrate that WBSCR22 is required for N⁷-methylation of G1639 in human 18S rRNA in vivo. Interestingly, the catalytic activity of WBSCR22 is not required for 18S pre-rRNA processing, suggesting that the key role of WBSCR22 in 40S subunit biogenesis is independent of its function as an RNA methyltransferase.     

Assembly of Functional Ribonucleoprotein Complexes by AU-rich Element RNA-binding Protein 1 (AUF1) Requires Base-dependent and -independent RNA Contacts

AU-rich element RNA-binding protein 1 (AUF1) regulates the stability and/or translational efficiency of diverse mRNA targets, including many encoding products controlling the cell cycle, apoptosis, and inflammation by associating with AU-rich elements residing in their 3′-untranslated regions. Previous biochemical studies showed that optimal AUF1 binding requires 33–34 nucleotides with a strong preference for U-rich RNA despite observations that few AUF1-associated cellular mRNAs contain such extended U-rich domains. Using the smallest AUF1 isoform (p37^AUF1) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37^AUF1 RNP complexes within a larger RNA context. In particular, p37^AUF1 binding to short AU-rich RNA targets was significantly stabilized by interactions with a 3′-purine residue and largely base-independent but non-ionic contacts 5′ of the AU-rich site. RNP stabilization by the upstream RNA domain was associated with an enhanced negative change in heat capacity consistent with conformational changes in protein and/or RNA components, and fluorescence resonance energy transfer-based assays demonstrated that these contacts were required for p37^AUF1 to remodel local RNA structure. Finally, reporter mRNAs containing minimal high affinity p37^AUF1 target sequences associated with AUF1 and were destabilized in a p37^AUF1-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs but also suggest how AUF1 binding could regulate protein and/or microRNA binding events at adjacent sites.     

m6A modification of HSATIII lncRNAs regulates temperature‐dependent splicing

Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress‐inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature‐dependent Ser/Arg‐rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA‐binding proteins revealed HSATIII association with proteins related to N⁶‐methyladenosine (m⁶A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m⁶A‐modified. nSBs sequester the m⁶A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m⁶A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m⁶A modification of pre‐mRNAs, leading to repression of m⁶A‐dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature‐dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m⁶A‐modified architectural lncRNAs.     

Expression of atrial natriuretic peptide receptor-A antagonizes the mitogen-activated protein kinases (Erk2 and P38MAPK) in cultured human vascular smooth muscle cells

To understand the signaling mechanisms of atrial natriuretic peptide (ANP) receptor-A (NPRA), we studied the effect of the ANP/NPRA system on mitogen-activated protein kinases (MAPKs), with particular emphasis on the extracellular-regulated kinase (Erk2) and stress-activated protein kinase (p38^MAPK) in cultured human vascular smooth muscle cells (HVSMC). Angiotensin II (ANG II) and platelet-derived growth factor (PDGF) stimulated the immunoreactive Erk2 and p38^MAPK activities and their protein levels by 2–4 fold. The pretreatment of cells with ANP significantly inhibited the agonist-stimulated Erk2 and p38^MAPK activities and protein expression by 65–75% in HVSMC transiently transfected with NPRA, as compared with only 18–22% inhibition in vector-transfected cells. The pretreatment of cells with KT5823, an inhibitor of cGMP-dependent protein kinase (PKG), reversed the inhibitory effects of ANP on MAPK activities and protein expression by 90–95%. PD98059, which inhibits Erk2 by directly inhibiting the MAPK-kinase (MEK), and SB202192, a selective antagonist of p38^MAPK, blocked the Erk2 and p38^MAPK activities, respectively. Interestingly, ANP stimulated the MAPK-phosphatase-3 (MKP-3) protein levels by more than 3-fold in HVSMC over-expressing NPRA, suggesting that ANP-dependent inhibition of MAPKs may also proceed by stimulating the phosphatase cascade. These present findings provide the evidence that ANP exerts inhibitory effects on agonist-stimulated MAPKs (Erk2 and p38^MAPK) activities and protein levels in a 2-fold manner: by antagonizing the upstream signaling pathways and by activation of MKP-3 to counter-regulate MAPKs in a cGMP and PKG-dependent manner. Our results identify a signal transduction pathway in HVSMC that could contribute to vascular remodeling and structural changes in human hypertension.     

Crystal Structure of the S. solfataricus Archaeal Exosome Reveals Conformational Flexibility in the RNA-Binding Ring

Background

The exosome complex is an essential RNA 3′-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry.

Methodology/Principal Findings

Here we report an asymmetric 2.9 Å Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation.

Conclusion/Significance

This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.