ncRNAseq: simple modifications to RNA-seq library preparation allow recovery and analysis of mid-sized non-coding RNAs

Despite their abundance, mid-sized RNAs (30–300 nt) have not been extensively studied by high-throughput sequencing, mostly due to selective loss in library preparation. The authors propose simple and inexpensive modifications to the Illumina TruSeq protocol (ncRNAseq), allowing the capture and sequencing of mid-sized non-coding RNAs without detriment to the coverage of coding mRNAs. This protocol is coupled with a two-step alignment: a pre-alignment to a curated non-coding genome, passing only the non-mapping reads to a standard genomic alignment. ncRNAseq correctly assigns the highest read-numbers to established abundant non-coding RNAs and correctly identifies cytosolic and nuclear enrichment of known non-coding RNAs in two cell lines.

The behavioral variant of Alzheimer's disease does not show a selective loss of Von Economo and phylogenetically related neurons in the anterior cingulate cortex

Background: The neurobiological origins of the early and predominant behavioral changes seen in the behavioral variant of Alzheimer's disease (bvAD) remain unclear. A selective loss of Von Economo Neurons (VENs) and phylogenetically related neurons have been observed in behavioral variant frontotemporal dementia (bvFTD) and several psychiatric diseases. Here, we assessed whether these specific neuronal populations show a selective loss in bvAD. Methods: VENs and GABA receptor subunit theta (GABRQ)-immunoreactive pyramidal neurons of the anterior cingulate cortex (ACC) were quantified in post-mortem tissue of patients with bvAD (n=9) and compared to typical AD (tAD, n=6), bvFTD due to frontotemporal lobar degeneration based on TDP-43 pathology (FTLD, n=18) and controls (n=13) using ANCOVAs adjusted for age and Bonferroni corrected. In addition, ratios of VENs and GABRQ-immunoreactive (GABRQ-ir) pyramidal neurons over all Layer 5 neurons were compared between groups to correct for overall Layer 5 neuronal loss. Results: The number of VENs or GABRQ-ir neurons did not differ significantly between bvAD (VENs: 26.0±15.3, GABRQ-ir pyramidal: 260.44±87.13) and tAD (VENs: 32.0±18.1, p=1.00, GABRQ-ir pyramidal: 349.83±109.64, p=0.38) and controls (VENs: 33.5±20.3, p=1.00, GABRQ-ir pyramidal: 339.38±95.88, p=0.37). Compared to bvFTD, patients with bvAD showed significantly more GABRQ-ir pyramidal neurons (bvFTD: 140.39±82.58, p=0.01) and no significant differences in number of VENs (bvFTD: 10.9±13.8, p=0.13). Results were similar when assessing the number of VENs and GABRQ-ir relative to all neurons of Layer 5. Discussion: VENs and phylogenetically related neurons did not show a selective loss in the ACC in patients with bvAD. Our results suggest that, unlike in bvFTD, the clinical presentation in bvAD may not be related to the loss of VENs and related neurons in the ACC.

Titin Circular RNAs Create a Back-Splice Motif Essential for SRSF10 Splicing

Background: TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. Methods: We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell–derived cardiomyocytes. Results: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell–derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as MEF2A and CASQ2 . Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. Conclusions: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation.

Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols

Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.

Disentangling ecological and taphonomic signals in ancient food webs

Analyses of ancient food webs reveal important paleoecological processes and responses to a range of perturbations throughout Earth's history, such as climate change. These responses can inform our forecasts of future biotic responses to similar perturbations. However, previous analyses of ancient food webs rarely accounted for key differences between modern and ancient community data, particularly selective loss of soft-bodied taxa during fossilization. To consider how fossilization impacts inferences of ancient community structure, we (1) analyzed node-level attributes to identify correlations between ecological roles and fossilization potential and (2) applied selective information loss procedures to food web data for extant systems. We found that selective loss of soft-bodied organisms has predictable effects on the trophic structure of “artificially fossilized” food webs because these organisms occupy unique, consistent food web positions. Fossilized food webs misleadingly appear less stable (i.e., more prone to trophic cascades), with less predation and an overrepresentation of generalist consumers. We also found that ecological differences between soft- and hard-bodied taxa—indicated by distinct positions in modern food webs—are recorded in an early Eocene web, but not in Cambrian webs. This suggests that ecological differences between the groups have existed for ≥48 Myr. Our results indicate that accounting for soft-bodied taxa is vital for accurate depictions of ancient food webs. However, the consistency of information loss trends across the analyzed food webs means it is possible to predict how the selective loss of soft-bodied taxa affects food web metrics, which can permit better modeling of ancient communities.