bims-micpro 2024-06-09 papers

Brief Bioinform. 2024 May 23. pii: bbae268. [Epub ahead of print]25(4):

Comparison of software packages for detecting unannotated translated small open reading frames by Ribo-seq.

Gregory Tong, Nasun Hah, Thomas F Martinez.

Accurate and comprehensive annotation of microprotein-coding small open reading frames (smORFs) is critical to our understanding of normal physiology and disease. Empirical identification of translated smORFs is carried out primarily using ribosome profiling (Ribo-seq). While effective, published Ribo-seq datasets can vary drastically in quality and different analysis tools are frequently employed. Here, we examine the impact of these factors on identifying translated smORFs. We compared five commonly used software tools that assess open reading frame translation from Ribo-seq (RibORFv0.1, RibORFv1.0, RiboCode, ORFquant, and Ribo-TISH) and found surprisingly low agreement across all tools. Only ~2% of smORFs were called translated by all five tools, and ~15% by three or more tools when assessing the same high-resolution Ribo-seq dataset. For larger annotated genes, the same analysis showed ~74% agreement across all five tools. We also found that some tools are strongly biased against low-resolution Ribo-seq data, while others are more tolerant. Analyzing Ribo-seq coverage revealed that smORFs detected by more than one tool tend to have higher translation levels and higher fractions of in-frame reads, consistent with what was observed for annotated genes. Together these results support employing multiple tools to identify the most confident microprotein-coding smORFs and choosing the tools based on the quality of the dataset and the planned downstream characterization experiments of the predicted smORFs.

Keywords: Ribo-seq; microprotein; smORF annotation; translation

DOI: https://doi.org/10.1093/bib/bbae268

J Proteomics. 2024 May 31. pii: S1874-3919(24)00146-5. [Epub ahead of print]303 105214

Identification of phosphorylated small ORF-encoded peptides in Hep3B cells by LC/MS/MS.

Mingbo Peng, Yutian Zhou, Cuihong Wan.

Small ORF-encoded peptides (SEPs) are a class of low molecular weight proteins and peptides comprising <100 amino acids with important functions in various life activities. Although the sequence length is short, SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. In this work, we enriched phosphopeptides with IMAC and TiO2 materials and analyzed the phosphorylated SEPs in Hep3B cells. A total of 24 phosphorylated SEPs were identified, and 11 SEPs were coded by ncRNA. For the sequence analysis, we found that the general characteristics of phosphorylated SEPs are roughly the same as canonical proteins. Besides, two phosphorylation SEPs have the Stathmin family signature 2 motif, which can regulate the microtubule cytoskeleton. Some SEPs have domains or signal peptides, indicating their specific functions and subcellular locations. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of some SEPs. However, only one-fifth of the predicted phosphorylation sites were identified by LC/MS/MS, indicating that many SEP PTMs are hidden in the dark, waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation. SIGNIFICANCE: Small ORF-encoded peptides (SEPs) are important in various life activities. Although the sequence length is short (<100AA), SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. We enriched phosphopeptides and analyzed the phosphorylated SEPs in Hep3B cells. That is the first time to explore the PTM of SPEs systematically. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of SEPs. More SEP PTMs are hidden in the dark and waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation.

Keywords: Kinases; Microprotein; PTM; Phosphorylation; Small ORF-encoded peptides

DOI: https://doi.org/10.1016/j.jprot.2024.105214

Cell Calcium. 2024 May 24. pii: S0143-4160(24)00068-X. [Epub ahead of print]121 102910

Phosphorylation of phospholamban promotes SERCA2a activation by dwarf open reading frame (DWORF).

Elisa Bovo, Thomas Jamrozik, Daniel Kahn, Patryk Karkut, Seth L Robia, Aleksey V Zima.

In cardiac myocytes, the type 2a sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) plays a key role in intracellular Ca regulation. Due to its critical role in heart function, SERCA2a activity is tightly regulated by different mechanisms, including micropeptides. While phospholamban (PLB) is a well-known SERCA2a inhibitor, dwarf open reading frame (DWORF) is a recently identified SERCA2a activator. Since PLB phosphorylation is the most recognized mechanism of SERCA2a activation during adrenergic stress, we studied whether PLB phosphorylation also affects SERCA2a regulation by DWORF. By using confocal Ca imaging in a HEK293 expressing cell system, we analyzed the effect of the co-expression of PLB and DWORF using a bicistronic construct on SERCA2a-mediated Ca uptake. Under these conditions of matched expression of PLB and DWORF, we found that SERCA2a inhibition by non-phosphorylated PLB prevails over DWORF activating effect. However, when PLB is phosphorylated at PKA and CaMKII sites, not only PLB's inhibitory effect was relieved, but SERCA2a was effectively activated by DWORF. Förster resonance energy transfer (FRET) analysis between SERCA2a and DWORF showed that DWORF has a higher relative affinity for SERCA2a when PLB is phosphorylated. Thus, SERCA2a regulation by DWORF responds to the PLB phosphorylation status, suggesting that DWORF might contribute to SERCA2a activation during conditions of adrenergic stress.

Keywords: Ca pump; Ca signaling; DWORF; HEK293 cells; Phospholamban

DOI: https://doi.org/10.1016/j.ceca.2024.102910

NAR Cancer. 2024 Jun;6(2): zcae026

Non-canonical mRNA translation initiation in cell stress and cancer.

Mélanie Mahé, Tiffany Rios-Fuller, Olga Katsara, Robert J Schneider.

The now well described canonical mRNA translation initiation mechanism of m7G 'cap' recognition by cap-binding protein eIF4E and assembly of the canonical pre-initiation complex consisting of scaffolding protein eIF4G and RNA helicase eIF4A has historically been thought to describe all cellular mRNA translation. However, the past decade has seen the discovery of alternative mechanisms to canonical eIF4E mediated mRNA translation initiation. Studies have shown that non-canonical alternate mechanisms of cellular mRNA translation initiation, whether cap-dependent or independent, serve to provide selective translation of mRNAs under cell physiological and pathological stress conditions. These conditions typically involve the global downregulation of canonical eIF4E1/cap-mediated mRNA translation, and selective translational reprogramming of the cell proteome, as occurs in tumor development and malignant progression. Cancer cells must be able to maintain physiological plasticity to acquire a migratory phenotype, invade tissues, metastasize, survive and adapt to severe microenvironmental stress conditions that involve inhibition of canonical mRNA translation initiation. In this review we describe the emerging, important role of non-canonical, alternate mechanisms of mRNA translation initiation in cancer, particularly in adaptation to stresses and the phenotypic cell fate changes involved in malignant progression and metastasis. These alternate translation initiation mechanisms provide new targets for oncology therapeutics development.

DOI: https://doi.org/10.1093/narcan/zcae026

Transl Res. 2024 Jun 03. pii: S1931-5244(24)00109-9. [Epub ahead of print]

LncRNA MFRL regulates the phenotypic switch of vascular smooth muscle cells to attenuate arterial remodeling by encoding a novel micropeptide MFRLP.

Xiaocong Liu, Siyu Chen, Wei Luo, Chen Yu, Shaohua Yan, Li Lei, Shifeng Qiu, Xinxin Lin, Ting Feng, Jinglin Shi, Qiuxia Zhang, Hongbin Liang, Xuewei Liu, Alex Pui-Wai Lee, Lei Zheng, Xinlu Zhang, Jiancheng Xiu.

BACKGROUND: Arterial remodeling is a common pathophysiological change in the pathogenesis of cardiovascular diseases in which the phenotypic switch of vascular smooth muscle cells (VSMC) plays an important role. Recently, an increasing number of long non-coding RNAs(lncRNAs) have been shown to encode micropeptides that play biological roles and have great clinical transformation potential. However, the role of micropeptides encoded by lncRNAs in arterial remodeling has not been well studied and requires further exploration.
METHODS AND RESULTS: Through bioinformatic analysis and experimental verification, we found that a new lncRNA, the mitochondrial function-related lncRNA (MFRL), encodes a 64-amino acid micropeptide, MFRLP. MFRL and MFRLP play important roles in the phenotypic switch of VSMC. Further experiments showed that MFRLP interacts with mitochondrial cytochrome b to reduce accumulation of reactive oxygen species, suppress mitophagy and inhibit the VSMC switch from contractile to synthetic phenotype.
CONCLUSIONS: LncRNA MFRL encodes the micropeptide MFRLP, which interacts with mitochondrial cytochrome b to inhibit the VSMC switch from contractile to synthetic phenotype and improve arterial remodeling.

Keywords: MT-CYTB; ROS; arterial remodeling; lncRNA; micropeptide; mitophagy

DOI: https://doi.org/10.1016/j.trsl.2024.05.009