Hyeshik Chang

Publication date: June 2014
Source:Trends in Biochemical Sciences, Volume 39, Issue 6
Author(s): Dinghai Zheng , Bin Tian
Global investigation of poly(A) tails has been hindered by technical challenges. In a recent advance, two groups developed deep sequencing methods to globally interrogate poly(A) tail length and sequence with high precision, opening new avenues for investigation of poly(A) tail functions in mRNA metabolism. Initial applications of these methods reveal insights into the relationship between poly(A) tail length and translational efficiency, and identify widespread uridylation and guanylation at the 3′ ends of transcripts.

Publication date: May 2014
Source:Trends in Genetics, Volume 30, Issue 5
Author(s): Andreas Wachter
The precise temporal and spatial coordination of gene activity, based on the integration of internal and external signals, is crucial for the accurate functioning of all biological processes. Although the basic principles of gene expression were established some 60 years ago, recent research has revealed a surprising complexity in the control of gene activity. Many of these gene regulatory mechanisms occur at the level of the mRNA, including sophisticated gene control tasks mediated by structured mRNA elements. We now know that mRNA folds can serve as highly specific receptors for various types of molecules, as exemplified by metabolite-binding riboswitches, and interfere with pro- and eukaryotic gene expression at the level of transcription, translation, and RNA processing. Gene regulation by structured mRNA elements comprises versatile strategies including self-cleaving ribozymes, RNA-folding-mediated occlusion or presentation of cis-regulatory sequences, and sequestration of trans-acting factors including other RNAs and proteins.

Caution required for handling genome editing technology.

Trends Biotechnol. 2014 May;32(5):234-7

Authors: Araki M, Nojima K, Ishii T

Abstract
Genome-editing technology, although a robust tool for genetic engineering, is creating indistinct regulatory boundaries between naturally occurring and modified organisms. However, researchers must act with caution in research and development to avoid misleading society. Furthermore, appropriate regulations should be proactively discussed and established for handling genome-editing technology.

PMID: 24767735 [PubMed - in process]

Findings may be having a profound impact on everything from animal behavior experiments to human clinical trials

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High-resolution genomic analysis of human mitochondrial RNA sequence variation.

Science. 2014 Apr 25;344(6182):413-5

Authors: Hodgkinson A, Idaghdour Y, Gbeha E, Grenier JC, Hip-Ki E, Bruat V, Goulet JP, de Malliard T, Awadalla P

Abstract
Mutations in the mitochondrial genome are associated with multiple diseases and biological processes; however, little is known about the extent of sequence variation in the mitochondrial transcriptome. By ultra-deeply sequencing mitochondrial RNA (>6000×) from the whole blood of ~1000 individuals from the CARTaGENE project, we identified remarkable levels of sequence variation within and across individuals, as well as sites that show consistent patterns of posttranscriptional modification. Using a genome-wide association study, we find that posttranscriptional modification of functionally important sites in mitochondrial transfer RNAs (tRNAs) is under strong genetic control, largely driven by a missense mutation in MRPP3 that explains ~22% of the variance. These results reveal a major nuclear genetic determinant of posttranscriptional modification in mitochondria and suggest that tRNA posttranscriptional modification may affect cellular energy production.

PMID: 24763589 [PubMed - in process]

While translational regulation is widely thought to contribute significantly to cellular differentiation programs, there are few genome-wide studies approaching this question. Here, the authors use ribosome profiling and mRNA-seq to assess gene-specific translation activity genome-wide in response to glucose starvation in yeast. Interestingly, a minority of transcripts comprise the majority of translating mRNA in both rapidly dividing and glucose-starved cells, though the identities of the translationally enriched mRNAs are distinct. Additionally, the authors identify features of the translational response that are specifically mediated by protein kinase A (PKA). These studies nicely reveal growth-state specialization of the "translatome."

An efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library is described. BC200 RNA was used as an RNA antigen. It is shown that an optimal binding antibody recognizes a domain of BC200 in a structure- and sequence-dependent manner and that, in breast cancer cells, the amount of antibody-recognizable RNA is not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms.

Mitochondrial posttranscriptional variation is common among humans and can be attributed to a nuclear gene. Authors: Alan Hodgkinson, Youssef Idaghdour, Elias Gbeha, Jean-Christophe Grenier, Elodie Hip-Ki, Vanessa Bruat, Jean-Philippe Goulet, Thibault de Malliard, Philip Awadalla

Sizing up the poly(A) tail: insights from deep sequencing.

Trends Biochem Sci. 2014 Apr 18;

Authors: Zheng D, Tian B

Abstract
Global investigation of poly(A) tails has been hindered by technical challenges. In a recent advance, two groups developed deep sequencing methods to globally interrogate poly(A) tail length and sequence with high precision, opening new avenues for investigation of poly(A) tail functions in mRNA metabolism. Initial applications of these methods reveal insights into the relationship between poly(A) tail length and translational efficiency, and identify widespread uridylation and guanylation at the 3' ends of transcripts.

PMID: 24751511 [PubMed - as supplied by publisher]

Nature Reviews Molecular Cell Biology 15, 298 (2014). doi:10.1038/nrm3797

Author: Kim Baumann

The CSR-1 RNAi pathway activates transcription at a genome-wide level by controlling RNA polymerase II directionality.

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A promoter-level mammalian expression atlas.

Nature. 2014 Mar 27;507(7493):462-70

Authors: FANTOM Consortium and the RIKEN PMI and CLST (DGT)

Abstract
Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

PMID: 24670764 [PubMed - indexed for MEDLINE]

Genome-wide Mapping of Cellular Protein-RNA Interactions Enabled by Chemical Crosslinking.

Genomics Proteomics Bioinformatics. 2014 Apr 16;

Authors: Li X, Song J, Yi C

Abstract
RNA-protein interactions influence many biological processes. Identifying the binding sites of RNA-binding proteins (RBPs) remains as one of the most fundamental and important challenges to the studies of such interactions. Capturing RNA and RBPs via chemical crosslinking allows stringent purification procedures that significantly remove the non-specific RNA and protein interactions. Two major types of chemical crosslinking strategies have been developed to date, i.e., UV-enabled crosslinking and enzymatic mechanism-based covalent capture. In this review, we compare such strategies and their current applications, with an emphasis on the technologies themselves rather than the biology that has been revealed. We hope such methods could benefit broader audience and also urge for the development of new methods to study RNA-RBP interactions.

PMID: 24747191 [PubMed - as supplied by publisher]

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MOV10 Is a 5' to 3' RNA Helicase Contributing to UPF1 mRNA Target Degradation by Translocation along 3' UTRs.

Mol Cell. 2014 Apr 8;

Authors: Gregersen LH, Schueler M, Munschauer M, Mastrobuoni G, Chen W, Kempa S, Dieterich C, Landthaler M

Abstract
RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5' to 3' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5' to 3' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3' UTRs.

PMID: 24726324 [PubMed - as supplied by publisher]

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Hepatic rRNA Transcription Regulates High-Fat-Diet-Induced Obesity.

Cell Rep. 2014 Apr 16;

Authors: Oie S, Matsuzaki K, Yokoyama W, Tokunaga S, Waku T, Han SI, Iwasaki N, Mikogai A, Yasuzawa-Tanaka K, Kishimoto H, Hiyoshi H, Nakajima Y, Araki T, Kimura K, Yanagisawa J, Murayama A

Abstract
Ribosome biosynthesis is a major intracellular energy-consuming process. We previously identified a nucleolar factor, nucleomethylin (NML), which regulates intracellular energy consumption by limiting rRNA transcription. Here, we show that, in livers of obese mice, the recruitment of NML to rRNA gene loci is increased to repress rRNA transcription. To clarify the relationship between obesity and rRNA transcription, we generated NML-null (NML-KO) mice. NML-KO mice show elevated rRNA level, reduced ATP concentration, and reduced lipid accumulation in the liver. Furthermore, in high-fat-diet (HFD)-fed NML-KO mice, hepatic rRNA levels are not decreased. Both weight gain and fat accumulation in HFD-fed NML-KO mice are significantly lower than those in HFD-fed wild-type mice. These findings indicate that rRNA transcriptional activation promotes hepatic energy consumption, which alters hepatic lipid metabolism. Namely, hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage.

PMID: 24746822 [PubMed - as supplied by publisher]

Background: Most cellular proteins function as part of stable protein complexes. We recently showed that around 38% of proteins associate with mRNAs that encode interacting proteins, reflecting the cotranslational formation of the complex between the bait protein and the nascent peptides encoded by the interacting mRNAs. Here we hypothesise that these cotranslational protein-mRNA associations can be used to predict protein-protein interactions. Results: We found that the fission yeast Exo2 protein, which encodes an exonuclease of the XRN1 family, coimmunoprecipitates with the eti1 mRNA, which codes for a protein of unknown function and uninformative sequence. Based on this protein-mRNA association, we predicted that the Exo2 and Eti1 protein are part of the same complex, and confirmed this hypothesis by coimmunoprecipitation and colocalization of the proteins. Similarly, we show that the cotranslational interaction between the Sty1 MAP kinase and the cip2 mRNA, which encodes an RNA-binding protein, predicts a complex between Sty1 and Cip2. Conclusions: Our results demonstrate that cotranslational protein-mRNA associations can be used to identify new components of protein complexes.

Lou et al. (1) report a technique for increased sensitivity of DNA sequencing that they call “circle sequencing.” The authors’ method involves circularizing single-stranded DNA and performing rolling-circle amplification (RCA). Multiple duplicates are thereby generated, and errors can be removed by comparing the sequences of the duplicates. Lou et al....

MHC class I peptides are products of endogenous cellular protein degradation. Their prompt presentation, after rapid degradation of their newly synthesized source proteins, is needed to alert the immune system during pathogen infection. A possible source for such rapidly degrading proteins can be defective ribosome products (DRiPs), which include polypeptides...

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Functional Genomic Analysis of Human Mitochondrial RNA Processing.

Cell Rep. 2014 Apr 16;

Authors: Wolf AR, Mootha VK

Abstract
Both strands of human mtDNA are transcribed in continuous, multigenic units that are cleaved into the mature rRNAs, tRNAs, and mRNAs required for respiratory chain biogenesis. We sought to systematically identify nuclear-encoded proteins that contribute to processing of mtRNAs within the organelle. First, we devised and validated a multiplex MitoString assay that quantitates 27 mature and precursor mtDNA transcripts. Second, we applied MitoString profiling to evaluate the impact of silencing each of 107 mitochondrial-localized, predicted RNA-binding proteins. With the resulting data set, we rediscovered the roles of recently identified RNA-processing enzymes, detected unanticipated roles of known disease genes in RNA processing, and identified new regulatory factors. We demonstrate that one such factor, FASTKD4, modulates the half-lives of a subset of mt-mRNAs and associates with mtRNAs in vivo. MitoString profiling may be useful for diagnosing and deciphering the pathogenesis of mtDNA disorders.

PMID: 24746820 [PubMed - as supplied by publisher]

The thermodynamic parameters for single nucleotide bulge loops with ambiguity to the exact position and identity of the bulge, of the type AG/U or CU/G, were determined. In-line structure probing of such bulge loops embedded in a hairpin indicates that the bulged nucleotide is the one positioned further from the hairpin loop, irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair.

Nature Nanotechnology. doi:10.1038/nnano.2014.73

Authors: Kyuwan Lee, Yi Cui, Luke P. Lee & Joseph Irudayaraj

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MYC and the Art of MicroRNA Maintenance.

Cold Spring Harb Perspect Med. 2014 Apr 15;

Authors: Psathas JN, Thomas-Tikhonenko A

Abstract
MYC is a noncanonical transcription factor that binds to thousands of genomic loci and affects >15% of the human transcriptome, with surprisingly little overlap between MYC-bound and -regulated genes. This discordance raises the question whether MYC chooses its targets based on their individual biological effects ("a la carte") or by virtue of belonging to a certain group of genes (on a "prix fixe" basis). This review presents evidence for a prix fixe, posttranscriptional model whereby MYC initially deregulates a select number of microRNAs. These microRNAs then target a broad spectrum of genes based solely on the presence in their 3' UTRs (untranslated regions) of distinct "seed" sequences. Existing evidence suggests that there are significant microRNA components to all key MYC-driven phenotypes, including cell-cycle progression, apoptosis, metabolism, angiogenesis, metastasis, stemness, and hematopoiesis. Furthermore, each of these cell-intrinsic and -extrinsic phenotypes is likely attributable to deregulation of multiple microRNA targets acting in different, yet frequently overlapping, pathways. The habitual targeting of multiple genes within the same pathway might account for the robustness and persistence of MYC-induced phenotypes.

PMID: 24737842 [PubMed - as supplied by publisher]

A long noncoding RNA regulates dendritic cell differentiation and function. Authors: Pin Wang, Yiquan Xue, Yanmei Han, Li Lin, Cong Wu, Sheng Xu, Zhengping Jiang, Junfang Xu, Qiuyan Liu, Xuetao Cao

by Yujia A. Chan, Maria J. Aristizabal, Phoebe Y. T. Lu, Zongli Luo, Akil Hamza, Michael S. Kobor, Peter C. Stirling, Philip Hieter

DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26^th International Conference on Yeast Genetics and Molecular Biology, August 2013.

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Elements and machinery of non-coding RNAs: toward their taxonomy.

EMBO Rep. 2014 Apr 14;

Authors: Hirose T, Mishima Y, Tomari Y

Abstract
Although recent transcriptome analyses have uncovered numerous non-coding RNAs (ncRNAs), their functions remain largely unknown. ncRNAs assemble with proteins and operate as ribonucleoprotein (RNP) machineries, formation of which is thought to be determined by specific fundamental elements embedded in the primary RNA transcripts. Knowledge about the relationships between RNA elements, RNP machinery, and molecular and physiological functions is critical for understanding the diverse roles of ncRNAs and may eventually allow their systematic classification or "taxonomy." In this review, we catalog and discuss representative small and long non-coding RNA classes, focusing on their currently known (and unknown) RNA elements and RNP machineries.

PMID: 24731943 [PubMed - as supplied by publisher]

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Methylation protects microRNAs from an AGO1-associated activity that uridylates 5' RNA fragments generated by AGO1 cleavage.

Proc Natl Acad Sci U S A. 2014 Apr 14;

Authors: Ren G, Xie M, Zhang S, Vinovskis C, Chen X, Yu B

Abstract
In plants, methylation catalyzed by HEN1 (small RNA methyl transferase) prevents microRNAs (miRNAs) from degradation triggered by uridylation. How methylation antagonizes uridylation of miRNAs in vivo is not well understood. In addition, 5' RNA fragments (5' fragments) produced by miRNA-mediated RNA cleavage can be uridylated in plants and animals. However, the biological significance of this modification is unknown, and enzymes uridylating 5' fragments remain to be identified. Here, we report that in Arabidopsis, HEN1 suppressor 1 (HESO1, a miRNA nucleotidyl transferase) uridylates 5' fragments to trigger their degradation. We also show that Argonaute 1 (AGO1), the effector protein of miRNAs, interacts with HESO1 through its Piwi/Argonaute/Zwille and PIWI domains, which bind the 3' end of miRNA and cleave the target mRNAs, respectively. Furthermore, HESO1 is able to uridylate AGO1-bound miRNAs in vitro. miRNA uridylation in vivo requires a functional AGO1 in hen1, in which miRNA methylation is impaired, demonstrating that HESO1 can recognize its substrates in the AGO1 complex. On the basis of these results, we propose that methylation is required to protect miRNAs from AGO1-associated HESO1 activity that normally uridylates 5' fragments.

PMID: 24733911 [PubMed - as supplied by publisher]

Sperm RNA carries marks of trauma

Nature 508, 7496 (2014). http://www.nature.com/doifinder/10.1038/508296a

Author: Virginia Hughes

Stress alters the expression of small RNAs in male mice and leads to depressive behaviours in later generations.

An Rrp6-like Protein Positively Regulates Noncoding RNA Levels and DNA Methylation in Arabidopsis.

Mol Cell. 2014 Apr 8;

Authors: Zhang H, Tang K, Qian W, Duan CG, Wang B, Zhang H, Wang P, Zhu X, Lang Z, Yang Y, Zhu JK

Abstract
Rrp6-mediated nuclear RNA surveillance tunes eukaryotic transcriptomes through noncoding RNA degradation and mRNA quality control, including exosomal RNA decay and transcript retention triggered by defective RNA processing. It is unclear whether Rrp6 can positively regulate noncoding RNAs and whether RNA retention occurs in normal cells. Here we report that AtRRP6L1, an Arabidopsis Rrp6-like protein, controls RNA-directed DNA methylation through positive regulation of noncoding RNAs. Discovered in a forward genetic screen, AtRRP6L1 mutations decrease DNA methylation independently of exosomal RNA degradation. Accumulation of Pol V-transcribed scaffold RNAs requires AtRRP6L1 that binds to RNAs in vitro and in vivo. AtRRP6L1 helps retain Pol V-transcribed RNAs in chromatin to enable their scaffold function. In addition, AtRRP6L1 is required for genome-wide Pol IV-dependent siRNA production that may involve retention of Pol IV transcripts. Our results suggest that AtRRP6L1 functions in epigenetic regulation by helping with the retention of noncoding RNAs in normal cells.

PMID: 24726328 [PubMed - as supplied by publisher]

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RNase III: Genetics and function; structure and mechanism.

Annu Rev Genet. 2013;47:405-31

Authors: Court DL, Gan J, Liang YH, Shaw GX, Tropea JE, Costantino N, Waugh DS, Ji X

Abstract
RNase III is a global regulator of gene expression in Escherichia coli that is instrumental in the maturation of ribosomal and other structural RNAs. We examine here how RNase III itself is regulated in response to growth and other environmental changes encountered by the cell and how, by binding or processing double-stranded RNA (dsRNA) intermediates, RNase III controls the expression of genes. Recent insight into the mechanism of dsRNA binding and processing, gained from structural studies of RNase III, is reviewed. Structural studies also reveal new cleavage sites in the enzyme that can generate longer 3' overhangs.

PMID: 24274754 [PubMed - indexed for MEDLINE]

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A change-point model for identifying 3'UTR switching by next-generation RNA sequencing.

Bioinformatics. 2014 Apr 11;

Authors: Wang W, Wei Z, Li H

Abstract
MOTIVATION: Next-generation RNA sequencing offers an opportunity to investigate transcriptome in an unprecedented scale. Recent studies have revealed widespread alternative polyadenalytion (APA) in eukaryotes, leading to various mRNA isoforms differing in their 3'UTR, through which, the stability, localization and translation of mRNA can be regulated. However, very few, if any, methods and tools are available for directly analyzing this special alternative RNA processing event. Conventional methods rely on annotation of polyadenylation sites; yet, such knowledge remains incomplete, and identification of polyA sites is still challenging. The goal of this article is to develop methods for detecting 3'UTR switching without any prior knowledge of polyA annotations.
RESULTS: We propose a change-point model based on a likelihood ratio test for detecting 3'UTR switching. We develop a directional testing procedure for identifying dramatic shortening or lengthening events in 3'UTR, while controlling mixed directional FDR at a nominal level. To our knowledge, this is the first approach to analyze 3'UTR switching directly without relying on any polyA annotations. Simulation studies and applications to two real datasets reveal that our proposed method is powerful, accurate and feasible for the analysis of next-generation RNA sequencing data.
CONCLUSIONS: The proposed method will fill a void among alternative RNA processing analysis tools for transciptome studies. It can help to obtain additional insights from RNA sequencing data by understanding gene regulation mechanisms through the analysis of 3'UTR switching.
AVAILABILITY AND IMPLEMENTATION: The software is implemented in Java and can be freely downloaded from http://utr.sourceforge.net/.
CONTACT: zhiwei@njit.edu; hongzhe@mail.med.upenn.edu.

PMID: 24728858 [PubMed - as supplied by publisher]

Background: Intron-derived long noncoding RNAs with snoRNA ends (sno-lncRNAs) are highly expressed from the imprinted Prader-Willi syndrome (PWS) region on human chromosome 15. However, sno-lncRNAs from other regions of the human genome or from other genomes have not yet been documented. Results: By exploring non-polyadenylated transcriptomes from human, rhesus and mouse, we have systematically annotated sno-lncRNAs expressed in all three species. In total, using available data from a limited set of cell lines, 19 sno-lncRNAs have been identified with tissue- and species-specific expression patterns. Although primary sequence analysis revealed that snoRNAs themselves are conserved from human to mouse, sno-lncRNAs are not. PWS region sno-lncRNAs are highly expressed in human and rhesus monkey, but are undetectable in mouse. Importantly, the absence of PWS region sno-lncRNAs in mouse suggested a possible reason why current mouse models fail to fully recapitulate pathological features of human PWS. In addition, a RPL13A region sno-lncRNA was specifically revealed in mouse embryonic stem cells, and its snoRNA ends were reported to influence lipid metabolism. Interestingly, the RPL13A region sno-lncRNA is barely detectable in human. We further demonstrated that the formation of sno-lncRNAs is often associated with alternative splicing of exons within their parent genes, and species-specific alternative splicing leads to unique expression pattern of sno-lncRNAs in different animals. Conclusions: Comparative transcriptomes of non-polyadenylated RNAs among human, rhesus and mouse revealed that the expression of sno-lncRNAs is species-specific and that their processing is closely linked to alternative splicing of their parent genes. This study thus further demonstrates a complex regulatory network of coding and noncoding parts of the mammalian genome.