MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

http://bartellab.wi.mit.edu/publication_reprints/Bartel_Cell09.pdf

MicroRNAs: Target Recognition and Regulatory Functions

Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets

MicroRNAs (miRNAs) are small RNAs that regulate the expression of complementary messenger RNAs. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. With miRNA roles identified in developmental timing, cell death, cell proliferation, haematopoiesis and patterning of the nervous system, evidence is mounting that animal miRNAs are more numerous, and their regulatory impact more pervasive, than was previously suspected.

The functions of animal microRNAs

Recent work has revealed the existence of a class of small non-coding RNA species, known as microRNAs (miRNAs), which have critical functions across various biological processes. Here we use a new, bead-based flow cytometric miRNA expression profiling method to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers. The miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours. We observe a general downregulation of miRNAs in tumours compared with normal tissues. Furthermore, we were able to successfully classify poorly differentiated tumours using miRNA expression profiles, whereas messenger RNA profiles were highly inaccurate when applied to the same samples. These findings highlight the potential of miRNA profiling in cancer diagnosis.

/pdf/microrna-expression-profiles-classify-human-cancers-43jqfv3rwu.pdf

MicroRNA expression profiles classify human cancers

Abstract Eukaryotic precursor mRNAs often harbor noncoding introns that must be removed prior to translation. Accurate splicing of precursor messenger RNA depends on placement and assembly of small nuclear ribonucleoprotein (snRNP) sub-complexes of the spliceosome. Yeast (Saccharomyces cerevisiae) studies established a role in splice-site selection for PRE-RNA PROCESSING8 (PRP8), a conserved spliceosome scaffolding protein of the U5 snRNP. However, analogous splice-site selection studies in multicellular eukaryotes are lacking. Such studies are crucial for a comprehensive understanding of alternative splicing, which is extensive in plants and animals but limited in yeast. In this work, we describe an Arabidopsis (Arabidopsis thaliana) prp8a mutant that modulates splice-site selection. We isolated prp8a-14 from a screen for suppressors of pex14-6, which carries a splice-site mutation in the PEROXIN14 (PEX14) peroxisome biogenesis gene. To elucidate Arabidopsis PRP8A function in spliceosome fidelity, we combined prp8a-14 with various pex14 splice-site mutations and monitored the double mutants for physiological and molecular consequences of dysfunctional and functional peroxisomes that correspond to impaired and recovered splicing, respectively. prp8a-14 restored splicing and PEX14 function to alleles with mutations in the exonic guanine of the 5′-splice site but did not restore splicing or function to alleles with mutations in the intronic guanine of 5′- or 3′-splice sites. We used RNA-seq to reveal the systemic impact of prp8a-14 and found hundreds of differentially spliced transcripts and thousands of transcripts with significantly altered levels. Among differentially spliced transcripts, prp8a-14 significantly altered 5′- and 3′-splice-site utilization to favor sites resulting in shorter introns. This study provides a genetic platform for probing splicing in plants and hints at a role for plant PRP8 in splice-site selection.

/pdf/an-arabidopsis-pre-rna-processing8a-prp8a-missense-allele-bj7wfxvb.pdf

An Arabidopsis pre-RNA processing8a (prp8a) missense allele restores splicing of a subset of mis-spliced mRNAs

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Joanne Chory



AS a senior graduate student contemplating my postdoctoral options, I was struck by the paper “ Arabidopsis thaliana mutant that develops as a light-grown plant in the absence of light” ([Chory et al. 1989][2]). The temerity of looking for mutant plants that

https://www.genetics.org/content/genetics/191/2/297.full.pdf

The 2012 Genetics Society of America Medal: Joanne Chory.

Lipid droplets are organelles conserved across eukaryotes that store and release neutral lipids to regulate energy homeostasis. In oilseed plants, fats stored in seed lipid droplets provide fixed carbon for seedling growth before photosynthesis begins. As fatty acids released from lipid droplet triacylglycerol are catabolized in peroxisomes, lipid droplet coat proteins are ubiquitinated, extracted, and degraded. In Arabidopsis seeds, the predominant lipid droplet coat protein is OLEOSIN1 (OLE1). To identify genes modulating lipid droplet dynamics, we mutagenized a line expressing mNeonGreen-tagged OLE1 expressed from the OLE1 promoter and isolated mutants with delayed oleosin degradation. From this screen, we identified four miel1 mutant alleles. MIEL1 (MYB30-interacting E3 ligase 1) targets specific MYB transcription factors for degradation during hormone and pathogen responses [D. Marino et al., Nat. Commun. 4, 1476 (2013); H. G. Lee and P. J. Seo, Nat. Commun. 7, 12525 (2016)] but had not been implicated in lipid droplet dynamics. OLE1 transcript levels were unchanged in miel1 mutants, indicating that MIEL1 modulates oleosin levels posttranscriptionally. When overexpressed, fluorescently tagged MIEL1 reduced oleosin levels, causing very large lipid droplets. Unexpectedly, fluorescently tagged MIEL1 localized to peroxisomes. Our data suggest that MIEL1 ubiquitinates peroxisome-proximal seed oleosins, targeting them for degradation during seedling lipid mobilization. The human MIEL1 homolog (PIRH2; p53-induced protein with a RING-H2 domain) targets p53 and other proteins for degradation and promotes tumorigenesis [A. Daks et al., Cells 11, 1515 (2022)]. When expressed in Arabidopsis, human PIRH2 also localized to peroxisomes, hinting at a previously unexplored role for PIRH2 in lipid catabolism and peroxisome biology in mammals.

Bonnie Bartel

Papers

An Arabidopsis pre-RNA processing8a (prp8a) missense allele restores splicing of a subset of mis-spliced mRNAs

The 2012 Genetics Society of America Medal: Joanne Chory.

The ubiquitin-protein ligase MIEL1 localizes to peroxisomes to promote seedling oleosin degradation and lipid droplet mobilization.