bims-micpro 2021-02-21 papers

J Biol Chem. 2021 Feb 10. pii: S0021-9258(21)00184-8. [Epub ahead of print] 100412

The transmembrane peptide DWORF activates SERCA2a via dual mechanisms.

Ang Li, Samantha L Yuen, Daniel R Stroik, Evan Kleinboehl, Razvan L Cornea, David D Thomas.

The Ca-ATPase isoform 2a (SERCA2a) pumps cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. Abnormally high cytosolic [Ca2+] is a central factor in heart failure, suggesting that augmentation of SERCA2a Ca2+ transport activity could be a promising therapeutic approach. SERCA2a is inhibited by the protein phospholamban (PLB), and a novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing PLB from binding to SERCA2a. However, establishing DWORF's precise physiological role requires further investigation. In the present study, we developed cell-based FRET biosensor systems that can report on protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the SERCA2a binding site, we transiently transfected DWORF into a stable HEK cell line expressing SERCA2a labeled with a FRET donor and PLB labeled with a FRET acceptor. We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Surprisingly, we also found that DWORF also activates SERCA's enzymatic activity directly in the absence of PLB at subsaturating calcium levels. Using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues P15 and W22 as necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

Keywords: FRET; biosensor; calcium ATPase; cardiac muscle; fluorescence resonance energy transfer

DOI: https://doi.org/10.1016/j.jbc.2021.100412

FEBS J. 2021 Feb 17.

Revisiting sORFs: overcoming challenges to identify and characterize functional microproteins.

Dörte Schlesinger, Simon J Elsässer.

Short open reading frames (sORFs), i.e. occurrences of a start and stop codon within 100 codons or less, can be found in organisms of all domains of life, outnumbering annotated protein-coding open reading frames (ORFs) by orders of magnitude. Even though functional proteins smaller than 100 amino acids are known, the coding potential of sORFs has often been overlooked, as it is not trivial to predict and test for functionality within the large number of sORFs. Recent advances in ribosome profiling and mass spectrometry approaches, together with refined bioinformatic predictions, have enabled a huge leap forward in this field and identified thousands of likely coding sORFs. A relatively low number of small proteins or microproteins produced from these sORFs have been characterized so far on the molecular, structural and/or mechanistic level. These however display versatile and, in some cases, essential cellular functions, allowing for the exciting possibility that many more, previously unknown small proteins might be encoded in the genome, waiting to be discovered. This review will give an overview of the steadily growing microprotein field, focusing on eukaryotic small proteins. We will discuss emerging themes in the molecular action of microproteins, as well as advances and challenges in microprotein identification and characterization.

Keywords: Micropeptides; Microproteins; SEPs; sORFs; small proteins; translation

DOI: https://doi.org/10.1111/febs.15769

Cell Mol Life Sci. 2021 Jan 28.

The role of micropeptides in biology.

Rui Vitorino, Sofia Guedes, Francisco Amado, Manuel Santos, Nobuyoshi Akimitsu.

Micropeptides are small polypeptides coded by small open-reading frames. Progress in computational biology and the analyses of large-scale transcriptomes and proteomes have revealed that mammalian genomes produce a large number of transcripts encoding micropeptides. Many of these have been previously annotated as long noncoding RNAs. The role of micropeptides in cellular homeostasis maintenance has been demonstrated. This review discusses different types of micropeptides as well as methods to identify them, such as computational approaches, ribosome profiling, and mass spectrometry.

Keywords: Long noncoding RNA; Short ORF; Upstream ORF; sORF; uORF

DOI: https://doi.org/10.1007/s00018-020-03740-3

Elife. 2021 Feb 17. pii: e59303. [Epub ahead of print]10

One-shot analysis of translated mammalian lncRNAs with AHARIBO.

Luca Minati, Claudia Firrito, Alessia Del Piano, Alberto Peretti, Simone Sidoli, Daniele Peroni, Romina Belli, Francesco Gandolfi, Alessandro Romanel, Paola Bernabo, Jacopo Zasso, Alessandro Quattrone, Graziano Guella, Fabio Lauria, Gabriella Viero, Massimiliano Clamer.

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 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 lncRNAs.

Keywords: cell biology; human

DOI: https://doi.org/10.7554/eLife.59303

Nat Commun. 2021 02 17. 12(1): 1076

Determinants of genome-wide distribution and evolution of uORFs in eukaryotes.

Hong Zhang, Yirong Wang, Xinkai Wu, Xiaolu Tang, Changcheng Wu, Jian Lu.

Upstream open reading frames (uORFs) play widespread regulatory functions in modulating mRNA translation in eukaryotes, but the principles underlying the genomic distribution and evolution of uORFs remain poorly understood. Here, we analyze ~17 million putative canonical uORFs in 478 eukaryotic species that span most of the extant taxa of eukaryotes. We demonstrate how positive and purifying selection, coupled with differences in effective population size (Ne), has shaped the contents of uORFs in eukaryotes. Besides, gene expression level is important in influencing uORF occurrences across genes in a species. Our analyses suggest that most uORFs might play regulatory roles rather than encode functional peptides. We also show that the Kozak sequence context of uORFs has evolved across eukaryotic clades, and that noncanonical uORFs tend to have weaker suppressive effects than canonical uORFs in translation regulation. This study provides insights into the driving forces underlying uORF evolution in eukaryotes.

DOI: https://doi.org/10.1038/s41467-021-21394-y