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A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.

Cold stenothermal insects living in glacier-fed streams are stressed by temperature variations resulting from glacial retreat during global warming. The molecular aspects of insect response to environmental stresses remain largely unexplored. The aim of this study was to expand our knowledge of how a cold stenothermal organism controls gene expression at the transcriptional, translational, and protein level under warming conditions. Using the chironomid Diamesa tonsa as target species and a combination of RACE, qPCR, polysomal profiling, western blotting, and bioinformatics techniques, we discovered a new molecular pathway leading to previously overlooked adaptive strategies to stress. We obtained and characterized the complete cDNA sequences of three heat shock inducible 70 (hsp70) and two members of heat-shock cognate 70 (hsc70). Strikingly, we showed that a novel pseudo-hsp70 gene encoding a putative long noncoding RNA (lncRNA) which is transcribed during thermal stress, acting as a ribosome sponge to provide post-transcriptional control of HSP70 protein levels. The expression of the pseudo-hsp70 gene and its function suggest the existence of a new and unexpected mechanism to cope with thermal stress: lowering the pace of protein production to save energy and optimize resources for recovery.

Summary: Genetic alterations impacting ubiquitously expressed proteins involved in RNA metabolism often result in neurodegenerative conditions, with increasing evidence suggesting that translation defects can contribute to disease. Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, whose role in pathogenesis remains unclear. Here, we identified in vivo and in vitro translation defects that are cell autonomous and SMN dependent. By determining in parallel the in vivo transcriptome and translatome in SMA mice, we observed a robust decrease in translation efficiency arising during early stages of disease. We provide a catalogue of RNAs with altered translation efficiency, identifying ribosome biology and translation as central processes affected by SMN depletion. This was further supported by a decrease in the number of ribosomes in SMA motor neurons in vivo. Overall, our findings suggest ribosome biology as an important, yet largely overlooked, factor in motor neuron degeneration. : Bernabò et al. analyzed translation in a mouse model of spinal muscular atrophy and identified translation defects that arise early in pathogenesis, are cell autonomous, and are accompanied by a low number of axonal ribosomes in diseased nerves. Their findings highlight ribosome biology as a central hallmark of the disease. Keywords: neurodegeneration, motor neuron disease, ribosome, translatome, polysomal profiling, spinal muscular atrophy, SMN

Summary: Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution. : Clamer et al. present RiboLace, a method for isolating active ribosomes and associated proteins, intact mRNAs, or ribosome-protected fragments. RiboLace accurately quantifies translation levels, providing positional data of active ribosomes with nucleotide resolution. Requiring lower input than current ribosome profiling protocols, RiboLace can be used with challenging biological samples. Keywords: ribosome profiling, translation, puromycin, translational control, protein synthesis, ribosome, proteome, polysomal profiling, translatome

Ribosome profiling is a powerful technique used to study translation at the genome-wide level, generating unique information concerning ribosome positions along RNAs. Optimal localization of ribosomes requires the proper identification of the ribosome P-site in each ribosome protected fragment, a crucial step to determine the trinucleotide periodicity of translating ribosomes, and draw correct conclusions concerning where ribosomes are located. To determine the P-site within ribosome footprints at nucleotide resolution, the precise estimation of its offset with respect to the protected fragment is necessary. Here we present riboWaltz, an R package for calculation of optimal P-site offsets, diagnostic analysis and visual inspection of ribosome profiling data. Compared to existing tools, riboWaltz shows improved accuracies for P-site estimation and neat ribosome positioning in multiple case studies. riboWaltz was implemented in R and is available as an R package at https://github.com/LabTranslationalArchitectomics/RiboWaltz.

Acute toxicity and genotoxic activity of 11 pollutants were investigated in wild populations of Diamesa cinerella and Diamesa zernyi (Diptera Chironomidae) from two alpine streams (Italian Alps). D. cinerella was collected in two sites on the non-glacial Vermigliana stream, 50 m-upstream and 5-m downstream of the Wastewater Treatment Plant (WTP) at the Tonal Pass (1799 m a.s.l.). D. zernyi was collected in the Presena glacial stream, close to the glacier snout (2685 m a.s.l.). IV-instar larvae were exposed for 24-96 h to increasing concentrations of three drugs (ibuprofen-IBU, furosemide-FUR, trimethoprim-TMP), three personal care products (triclocarban-TCC, tonalid-TON, sucralose-SUCR), and five pesticides (boscalid-BOS, captan-CAP, chlorpyrifos-CPS, metolachlor-MET, terbuthylazine-TER). The experimental concentrations were from one to several million times higher than the highest environmental concentration (EC) measured in the study sites. Two mixtures of pesticides were also prepared: MIX 1K =103 x EC of CPS, MET and TER, and MIX 10K = 104 x EC of CPS, MET and TER. Species- and site-specific responses were observed for both tests. On the basis of survival data, both species resulted very resistant to pharmaceuticals (mainly to FUR for which no effects on survival and movement or pupation were observed), and more sensitive to pesticides (mainly to CPS, MET and CAP). Genotoxicity tests (Comet assay) highlighted a WTP effect under natural conditions and a genotoxic effect for 9 of the 11 tested compounds. Overall, a clear gradient of increasing resistance in larvae from the least (PR0) to the most polluted (TP_dw) site was highlighted by both tests, ecotoxicological and of genotoxicity, as also expected according to species autecology (D. zernyi is restricted to very cold and pristine habitats). D. cinerella living downstream of the effluent accumulates a significantly higher DNA damage than the other populations, highlighting a basal physiological stress condition in nature. It is plausible that these larvae possess chemical resistance strategies to survive already under natural conditions. Diamesa spp. exhibited a higher toxic resistance than any other model species tested to date under the same pollutants, probably associable to its strong cold resistance. The results emphasised that the measured concentrations of Contaminants of Emerging Concern (CECs) and pesticides seem to be far below those required to cause acute effects. However, the effects on freshwater communities of prolonged exposure to mixture of trace CECs and pesticides remain unknown.