Efficient 3'-pairing renders microRNA targeting less sensitive to mRNA seed accessibility.

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2-g8 in the miRNA), but pairing beyond the seed (3'-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3'-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3'-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3'-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3'-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3'-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3'-pairing. We propose that these results could refine predictions of effective target sites.

Importance of residue 248 in Escherichia coli RNase P RNA mediated cleavage.

tRNA genes are transcribed as precursors and RNase P generates the matured 5' end of tRNAs. It has been suggested that residue - 1 (the residue immediately 5' of the scissile bond) in the pre-tRNA interacts with the well-conserved bacterial RNase P RNA (RPR) residue A248 (Escherichia coli numbering). The way A248 interacts with residue - 1 is not clear. To gain insight into the role of A248, we analyzed cleavage as a function of A248 substitutions and N-1 nucleobase identity by using pre-tRNA and three model substrates. Our findings are consistent with a model where the structural topology of the active site varies and depends on the identity of the nucleobases at, and in proximity to, the cleavage site and their potential to interact. This leads to positioning of Mg2+ that activates the water that acts as the nucleophile resulting in efficient and correct cleavage. We propose that in addition to be involved in anchoring the substrate the role of A248 is to exclude bulk water from access to the amino acid acceptor stem, thereby preventing non-specific hydrolysis of the pre-tRNA. Finally, base stacking is discussed as a way to protect functionally important base-pairing interactions from non-specific hydrolysis, thereby ensuring high fidelity during RNA processing and the decoding of mRNA.

RNA:RNA interaction in ternary complexes resolved by chemical probing.

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain.

The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C- and S-domains between its loop and the P8 helix in the S-domain (the P8/ P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/ P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/ P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent.

Human neuromuscular aging: Sex differences revealed at the myocellular level.

Age-related muscle loss (sarcopenia) is a major clinical problem affecting both men and women - accompanied by muscle weakness, dysfunction, disability, and impaired quality of life. Current definitions of sarcopenia do not fully encompass the age-related changes in skeletal muscle. We therefore examined the influence of aging and sex on elements of skeletal muscle health using a thorough histopathological analysis of myocellular aging and assessments of neuromuscular performance. Two-hundred and twenty-one untrained males and females were separated into four age cohorts [mean age 25 y (n = 47), 37 y (n = 79), 61 y (n = 51), and 72 y (n = 44)]. Total (-12%), leg (-17%), and arm (-21%) lean mass were lower in both 61 y and 72 y than in 25 y or 37 y (P < 0.05). Knee extensor strength (-34%) and power (-43%) were lower (P < 0.05) in the older two groups, and explosive sit-to-stand power was lower by 37 y (P < 0.05). At the histological/myocellular level, type IIx atrophy was noted by 37 y and type IIa atrophy by 61 y (P < 0.05). These effects were driven by females, noted by substantial and progressive type IIa and IIx atrophy across age. Aged female muscle displayed greater within-type myofiber size heterogeneity and marked type I myofiber grouping (~5-fold greater) compared to males. These findings suggest the predominant mechanisms leading to whole muscle atrophy differ between aging males and females: myofiber atrophy in females vs. myofiber loss in males. Future studies will be important to better understand the mechanisms underlying sex differences in myocellular aging and optimize exercise prescriptions and adjunctive treatments to mitigate or reverse age-related changes.

Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements.

Two broad classes of RNase P trim the 5' leader of precursor tRNAs (pre-tRNAs): ribonucleoprotein (RNP)- and proteinaceous (PRORP)-variants. These two RNase P types, which use different scaffolds for catalysis, reflect independent evolutionary paths. While the catalytic RNA-based RNP form is present in all three domains of life, the PRORP family is restricted to eukaryotes. To obtain insights on substrate recognition by PRORPs, we examined the 5' processing ability of recombinant Arabidopsis thaliana PRORP1 (AtPRORP1) using a panel of pre-tRNASer variants and model hairpin-loop derivatives (pATSer type) that consist of the acceptor-T-stem stack and the T-/D-loop. Our data indicate the importance of the identity of N-1 (the residue immediately 5' to the cleavage site) and the N-1:N+73 base pair for cleavage rate and site selection of pre-tRNASer and pATSer. The nucleobase preferences that we observed mirror the frequency of occurrence in the complete suite of organellar pre-tRNAs in eight algae/plants that we analyzed. The importance of the T-/D-loop in pre-tRNASer for tight binding to AtPRORP1 is indicated by the 200-fold weaker binding of pATSer compared to pre-tRNASer, while the essentiality of the T-loop for cleavage is reflected by the near-complete loss of activity when a GAAA-tetraloop replaced the T-loop in pATSer. Substituting the 2'-OH at N-1 with 2'-H also resulted in no detectable cleavage, hinting at the possible role of this 2'-OH in coordinating Mg2+ ions critical for catalysis. Collectively, our results indicate similarities but also key differences in substrate recognition by the bacterial RNase P RNP and AtPRORP1: while both forms exploit the acceptor-T-stem stack and the elbow region in the pre-tRNA, the RNP form appears to require more recognition determinants for cleavage-site selection.

Heightened muscle inflammation susceptibility may impair regenerative capacity in aging humans.

The regenerative response of skeletal muscle to mechanically induced damage is impaired with age. Previous work in our laboratory suggests this may result from higher proinflammatory signaling in aging muscle at rest and/or a greater inflammatory response to damage. We, therefore, assessed skeletal muscle proinflammatory signaling at rest and 24 h after unaccustomed, loaded knee extension contractions that induced modest muscle damage (72% increase in serum creatine kinase) in a cohort of 87 adults across three age groups (AGE40, AGE61, and AGE76). Vastus lateralis muscle gene expression and protein cell signaling of the IL-6 and TNF-α pathways were determined by quantitative PCR and immunoblot analysis. For in vitro studies, cell signaling and fusion capacities were compared among primary myoblasts from young (AGE28) and old (AGE64) donors treated with TNF-α. Muscle expression was higher (1.5- to 2.1-fold) in AGE76 and AGE61 relative to AGE40 for several genes involved in IL-6, TNF-α, and TNF-like weak inducer of apoptosis signaling. Indexes of activation for the proinflammatory transcription factors signal transducer and activator of transcription-3 and NF-κB were highest in AGE76. Resistance loading reduced gene expression of IL-6 receptor, muscle RING finger 1, and atrogin-1, and increased TNF-like weak inducer of apoptosis receptor expression. Donor myoblasts from AGE64 showed impaired differentiation and fusion in standard media and greater NF-κB activation in response to TNF-α treatment (compared with AGE28). We show for the first time that human aging is associated with muscle inflammation susceptibility (i.e., higher basal state of proinflammatory signaling) that is present in both tissue and isolated myogenic cells and likely contributes to the impaired regenerative capacity of skeletal muscle in the older population.

Modulation of the dystrophin-associated protein complex in response to resistance training in young and older men.

The dystrophin-associated protein complex (DAPC) is a scaffold of proteins linking the intracellular cytoskeleton with the extracellular matrix that is integral to structural stability and integrity, signaling and mechanotransduction, and force transmission. We hypothesized that the expression of DAPC component proteins would be altered by resistance loading during progressive resistance training (PRT)-mediated myofiber hypertrophy, and we investigated whether aging influenced these changes. Seventeen young (27 yr) and 13 older (65 yr) men completed 16 wk of PRT with muscle biopsies at baseline (T1), 24 h after bout 1 (T2), and 24 h after the final bout at week 16 (T3). Myofiber hypertrophy in the young (type I 31%, P < 0.005; type II 40%, P < 0.001) far exceeded hypertrophy in the old (type II only, 19.5%, P < 0.05). PRT altered protein expression for caveolin-3 (decreased 24% by T3, P < 0.01), alpha(1)-syntrophin (increased 16% by T3, P < 0.05), alpha-dystrobrevin (fell 23% from T2 to T3, P < 0.01), and dystrophin [rose acutely (30% by T2, P < 0.05) and returned to baseline by T3]. The phosphorylation state of membrane neuronal nitric oxide synthase (Ser(1417)) decreased 70% (P < 0.005) by T3, particularly in the old (81%), whereas p38 MAPK phosphorylation increased twofold by T3 in the old (P < 0.01). We conclude that component proteins of the DAPC are modulated by PRT, which may serve to improve both structural and signaling functions during load-mediated myofiber hypertrophy. The blunted hypertrophic adaptation seen in old vs. young men may have resulted from overstress, as suggested by marked p38 MAPK activation in old men only.

Efficacy of myonuclear addition may explain differential myofiber growth among resistance-trained young and older men and women.

Skeletal muscle stem (satellite) cells supporting growth/regeneration are thought to be activated and incorporated into growing myofibers by both endocrine and locally expressed autocrine/paracrine growth factors, the latter being load sensitive. We recently found that myofiber hypertrophy with resistance training is superior in young men (YM) vs. young women and older adults (Kosek DJ, Kim JS, Petrella JK, Cross JM, and Bamman MM. J Appl Physiol 101: 531-544, 2006). We hypothesized that the advanced myofiber hypertrophy in YM is facilitated by myonuclear addition in response to a milieu promoting stem cell activation. Twenty-six young (27.0 +/- 1 yr, 50% women) and 26 older (63.7 +/- 1 yr, 50% women) adults completed 16 wk of knee extensor resistance training. Vastus lateralis biopsies were obtained at baseline, 24 h after one bout, and after 16 wk. Muscle stem cells were identified immunohistochemically with anti-neural cell adhesion molecule (NCAM+). Muscle transcript levels of IGF-I and mechanogrowth factor (MGF) were determined by RT-PCR. Serum IGF-I, IGF-binding protein (IGFBP)-3, IGFBP-1, total and free testosterone, sex hormone-binding globulin (SHBG), and androstenedione were assessed by radioimmunoassay. Myofiber hypertrophy was twofold greater in YM vs. others, and only YM increased NCAM+ cells per 100 myofibers (49%) and myonuclei per fiber (19%) (P < 0.05). IGF-IEa mRNA was higher in young and increased acutely (29%) with summation by 16 wk (96%) (P < 0.05). MGF mRNA increased only in young after one bout (81%) and by 16 wk (85%) (P < 0.001). Circulating IGF-I was twofold higher in young, whereas IGFBP-1 was lowest in YM (P < 0.05). Among men, free testosterone was 59% higher in YM (P < 0.01). Myonuclear addition was most effectively accomplished in YM, which likely drove the superior growth.

Efficacy of 3 days/wk resistance training on myofiber hypertrophy and myogenic mechanisms in young vs. older adults.

Resistance training (RT) has shown the most promise in reducing/reversing effects of sarcopenia, although the optimum regime specific for older adults remains unclear. We hypothesized myofiber hypertrophy resulting from frequent (3 days/wk, 16 wk) RT would be impaired in older (O; 60-75 yr; 12 women, 13 men), sarcopenic adults compared with young (Y; 20-35 yr; 11 women, 13 men) due to slowed repair/regeneration processes. Myofiber-type distribution and cross-sectional area (CSA) were determined at 0 and 16 wk. Transcript and protein levels of myogenic regulatory factors (MRFs) were assessed as markers of regeneration at 0 and 24 h postexercise, and after 16 wk. Only Y increased type I CSA 18% (P < 0.001). O showed smaller type IIa (-16%) and type IIx (-24%) myofibers before training (P < 0.05), with differences most notable in women. Both age groups increased type IIa (O, 16%; Y, 25%) and mean type II (O, 23%; Y, 32%) size (P < 0.05). Growth was generally most favorable in young men. Percent change scores on fiber size revealed an age x gender interaction for type I fibers (P < 0.05) as growth among Y (25%) exceeded that of O (4%) men. Myogenin and myogenic differentiation factor D (MyoD) mRNAs increased (P < 0.05) in Y and O, whereas myogenic factor (myf)-5 mRNA increased in Y only (P < 0.05). Myf-6 protein increased (P < 0.05) in both Y and O. The results generally support our hypothesis as 3 days/wk training led to more robust hypertrophy in Y vs. O, particularly among men. However, this differential hypertrophy adaptation was not explained by age variation in MRF expression.

Resting and load-induced levels of myogenic gene transcripts differ between older adults with demonstrable sarcopenia and young men and women.

Regenerative capacity appears to be impaired in sarcopenic muscle. As local growth factors and myogenic regulatory factors (MRFs) modulate repair/regeneration responses after overload, we hypothesized that resistance loading (RL)-induced expression of MRFs and muscle IGF-I-related genes would be blunted in older (O) males (M) and females (F) with demonstrable sarcopenia vs. young (Y) adults. Y (20-35 yr, 10 YF, 10 YM) and O (60-75 yr, 9 OF, 9 OM) underwent vastus lateralis biopsy before and 24 h after knee extensor RL. Sarcopenia was assessed by cross-sectional area of type I, IIa, and IIx myofibers. Transcript levels were assessed by relative RT-PCR and analyzed by age x gender x load repeated-measures ANOVA. O were sarcopenic based on type II atrophy with smaller type IIa (P < 0.05) and IIx (P < 0.001) myofibers. Within-gender cross-sectional area differences were more marked in F (OF < YF: IIa 21%, IIx 42%). Load effects (P < 0.05) were seen for four of seven mRNAs as IGF-IEa (34%), myogenin (53%), and MyoD (20%) increased, and myf-6 declined 10%. Increased IGF-IEa was driven by O (48%) and/or M (43%). An age x gender x load interaction was found for MyoD (P < 0.05). An age x load interaction for type 1 IGF receptor (P < 0.05) was driven by a small increase in O (16%, P < 0.05). A gender x load interaction (P < 0.05) was noted for IGF binding protein-4. Age effects (P < 0.05) resulted from higher MyoD (54%), myf-5 (21%), and IGF binding protein-4 (17%) in O and were primarily localized to F at baseline (OF > YF; MyoD 94%, myf-5 47%, P < 0.05). We conclude that RL acutely increases mRNA expression of IGF-IEa and myogenin, which may promote growth/regeneration in both Y and O. Higher resting levels of MRFs in OF vs. YF suggest elevated basal regenerative activity in sarcopenic muscle of OF.

Ethics and scientific publication.

This article summarizes the major categories of ethical violations encountered during submission, review, and publication of scientific articles. We discuss data fabrication and falsification, plagiarism, redundant and duplicate publication, conflict of interest, authorship, animal and human welfare, and reviewer responsibility. In each section, pertinent historical background and citation of relevant regulations and statutes are provided. Furthermore, a specific case(s) derived from actual situations is(are) presented. These cases were chosen to highlight the complexities that investigators and journals must face when dealing with ethical issues. A series of discussion questions follow each case. It is our hope that by increasing education and awareness of ethical matters relevant to scientific investigation and publication, deviations from appropriate conduct will be reduced.