学术报告：
费聪（李林组）Lys29-Linked Nonproteolytic Poly-Ubiquitination of Axin by Smurf1
Negatively Regulates Wnt/β-Catenin Signaling
苟兰涛（刘默芳组）Mouse piRNAs, in complex with MIWI and CAF1, mediate
mRNA clearance in late stage of spermatogenesis
胡西旵（李伯良组）Suppression of HCC by inhibiting a specific cholesterol metabolic
pathway to increase cytotoxic oxysterols
黄骞（王恩多组）Identification of essential nucleotides in tRNALeu to its functions by
using a constructed yeast tRNALeu knockout strain
柳童斐（宋保亮组）Ablation of gp78 in liver improves hyperlipidemia and insulin
resistance by inhibiting SREBP to decrease lipid biosynthesis
牟世荣（惠静毅组）Regulation of pre-mRNA alternative splicing by a multifunctional
protein YB-1
谢盛松（张永莲组）Caput epididymis-specific Cre transgenic mouse model and
functions of miR-34a in spermatogenesis
殷庆飞（陈玲玲组）Long noncoding RNAs with snoRNA ends
张润瑞（徐国良组）A Positive Role of Tet1 DNA Dioxygenase in the Regulation of
AdultNeural Progenitor Cell Proliferation
赵雅（吴立刚组）microRNA serves as a mRNA surveillance system by eliminating
nonsense messages
朱长兰（程红组）Premature Termination Codons Are Recognized in the Nucleus in A
Reading-Frame Dependent Manner
Lys29-Linked Nonproteolytic Poly-Ubiquitination of Axin by Smurf1 Negatively
Regulates Wnt/β-Catenin Signaling
Abstract
Ubiquitination plays an important role in modulating protein functions. As a
C2-WW-HECT type ubiquitin ligase, Smurf1 regulates a number of signaling
pathways by promoting ubiquitin-dependent degradation of its targeted proteins. Here,
we disclosed a nonproteolytic role of Smurf1 in regulating the function of Axin
protein in Wnt/β-catenin signaling. Our data demonstrate that Smurf1 interacts with
Axin in a noncanonical pattern, where its WW domains are not required, and
ubiquitinates Axin via Lys29 (K29)-linked poly-ubiquitin chains. Smurf1-mediated
Axin poly-ubiquitination disrupts Axin-LRP6 interaction, attenuates Wnt-stimulated
LRP6 phosphorylation and inhibits Wnt/β-catenin signaling. Furthermore,
Smurf1-Axin interaction and Axin ubiquitination are attenuated in the G2/M phase of
cell cycle, resulting in an increase in the response to Wnt stimulation at that stage.
Thus, our study unraveled a previously unappreciated role of Smurf1 in regulating
protein functions in a cell cycle-dependent manner.
Mouse piRNAs, in complex with MIWI and CAF1, mediate mRNA clearance in
late stage of spermatogenesis
Lan-Tao Gou, Yun-Ping Hu, Peng Dai, Li-Gang Wu, En-Duo Wang, and
Mo-Fang Liu
State Key Laboratory of Molecular Biology–Graduate School of Chinese Academy of
Sciences, Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
A unique feature in late stages of male-germ cell development is the remarkable
nuclear morphological change and cytoplasm elimination. In mature sperm, little
mRNAs retain; however, how the diverse and massive unwanted mRNAs in late stage
of spermatids are eliminated during sperm maturation remains largely unexplored.
Here, we show that piRNAs, to form pi-ribonucleoproteins (piRNPs) complexes with
murine PIWI (MIWI) and deadenylase CAF1, induce mRNA degradation through
deadenylation by imperfectly base-pairing with the 3-untranslated region (UTR) of
mRNAs in mouse testes. Interestingly, we found that the deadenylation and
degradation of piRNA target mRNAs occurred in elongating spermatids, suggesting
that piRNPs complexes mediate mRNA clearance in late stage of spermatid
development. In support of this, inhibition of piRNPs complexes led to up-regulation
of all validated piRNA targets as well as stabilization of hundreds of predicted piRNA
target mRNAs in elongating spermatids. Thus, our results support a model in which
piRNAs utilize their enormous repertoire of targeting capacity to induce the
degradation of massive mRNA transcripts in elongating spermatids, providing a novel
mechanism for mRNA clearance in late stages of male-germ cell development.
Moreover, our findings also indicate novel function of piRNAs in regulating protein
genes in addition to acting as the vanguard of genome defence in mammalian germ
cells.
Suppression of HCC by inhibiting a specific cholesterol metabolic pathway to
increase cytotoxic oxysterols
Ming Lu1†, Xi-Han Hu1†, Qin Li1, Ying Xiong1, Guang-Jing Hu1, Jia-Jia Xu1, Xiao-Nan
Zhao1, Xi-Xiao Wei1, Catherine C. Y. Chang2, Yin-Kun Liu3, Fa-Jun Nan4, Jia Li4, Ta-Yuan
Chang2, Bao-Liang Song1* & Bo-Liang Li1*
1
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
2
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755,
U.S.A.
3Liver
Cancer Institute of Zhong Shan Hospital, Fudan University, Shanghai 200031, China
4National
Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
†These authors contributed equally to this work.
*To whom correspondence should be addressed. E-mail: blli@sibs.ac.cn or blsong@sibs.ac.cn
Liver plays central roles in cholesterol homeostasis. Hepatocellular carcinoma
(HCC) impairs certain liver functions, and results in disorders in many processes
including cholesterol metabolism. However, the link between disordered cholesterol
metabolism and HCC development is unclear. Oxysterols are metabolites of
cholesterol. The build up of unesterified oxysterols are cytotoxic to cells. Oxysterols
produced and secreted by all extrahepatic tissues are further metabolized in liver. Here
we report a HCC-specific cholesterol metabolic pathway that involves the induced
ACAT2 gene expression, needed to esterify oxysterols for their secretion, in
approximately 50% of advanced HCC. Inhibiting ACAT2 can specifically suppress
the growth of HCC cell lines and xenograft tumors by increasing intracellular
unesterified oxysterols. Further mechanistic studies reveal that HCC-linked promoter
hypomethylation is essential for epigenetic induction of ACAT2 gene. Taken together,
our findings imply that inhibiting ACAT2 can be used as a novel therapeutic strategy
for HCC and other human cancer treatments.
Identification of essential nucleotides in tRNALeu to its functions by using a
constructed yeast tRNALeu knockout strain
The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its
cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of
accuracy and efficiency. Crucial bases in tRNALeu to aminoacylation or editing
functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro
methods. In the present study, we constructed two Saccharomyces cerevisiae tRNALeu
knockout strains carrying deletions of the genes for tRNALeu(GAG) and
tRNALeu(UAG). Disrupting the single gene encoding tRNALeu(GAG) had no
phenotypic consequence when compared to the wild-type strain. While disrupting the
three genes for tRNALeu(UAG) had a lethal effect on the yeast strain, indicating that
tRNALeu(UAG) decoding capacity could not be compensated by another tRNALeu
isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of
tRNALeu(UAG), a selection to identify critical tRNALeu elements was performed. In
this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or
editing ability by leucyl-tRNA synthetase were identified. Full modified
tRNALeu(UAG) WT and mutants were purified from yeast transformants and the in
vitro experiments were performed. The results are identical with the in vivo ones.
Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in
vivo studies and identification of essential nucleotides of the tRNA.
Ablation of gp78 in liver improves hyperlipidemia and insulin resistance by
inhibiting SREBP to decrease lipid biosynthesis
Tong-Fei Liu1, Jing-Jie Tang1, Pei-Shan Li1, Yang Shen1, Jia-Gui Li§, Hong-Hua
Miao1, Bo-Liang Li1, * and Bao-Liang Song 1, *
1
The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
320 Yue-Yang Road, Shanghai 200031, China.
§Current address: Institut de Ge´ne´ tique et de Biologie Mole´ culaire et Cellulaire
and Institut Clinique de la Souris, BP10142, 67404 Illkirch Cedex, France.
Abstract
gp78 is a membrane-anchored ubiquitin ligase mediating the degradation of
HMG-CoA reductase (HMGCR) and Insig-1. As a rate-limiting enzyme in cholesterol
biosynthesis, HMGCR undergoes rapid sterol-promoted degradation. In contrast,
destruction of Insig-1 releases its inhibition on SREBP and stimulates the expression
of lipogenic genes. Thus, gp78 has opposite effects on lipid biosynthesis. We here
generated liver-specific gp78 knockout (L-gp78-/-) mice and showed that although the
degradation of HMGCR was blunted, SREBP was suppressed due to the elevation of
Insig-1/-2, and therefore the lipid biosynthesis was decreased. The L-gp78-/- mice
were protected from diet- /age- induced obesity and glucose intolerance. The livers of
L-gp78-/- mice produced more FGF21, which activated thermogenesis in brown
adipocytes and enhanced energy expenditure. Together, the major function of gp78 in
liver is regulating lipid biosynthesis through SREBP pathway. Ablation of gp78
decreases the lipid levels and increases FGF21, and is beneficial to patients with
metabolic diseases.
Regulation of pre-mRNA alternative splicing by a multifunctional protein YB-1
Shirong Mu, Wenjuan Wei, Monika Heiner, Lijuan Cao, Jingyi Hui
The human Y box-binding protein 1 (YB-1) is a member of the evolutionarily
conserved nucleic acid binding protein family, which exhibits multiple functions in
the regulation of transcription,
mRNA stability, and translation. It is mainly
localized in the cytoplasm, but is highly expressed in the nucleus of tumors,
particularly in breast cancer cells. A number of studies demonstrate its role in
malignant transformation. Recently, several lines of evidence indicate that YB-1 is a
spliceosome-associated protein and is involved in alternative splicing, but the
underlying mechanism has remained elusive. In this study, we defined both CAUC
and CACC as high-affinity binding motifs for YB-1 by SELEX and demonstrated that
these newly defined motifs function as splicing enhancers. Interestingly, on the
endogenous CD44 gene, YB-1 appears to mediate a network interaction to activate
exon v5 inclusion via multiple CAUC motifs in both the alternative exon and its
upstream polypyrimidine tract. U2AF65 is an essential splicing factor that recognizes
the polypyrimidine tracts in the 3’ splice sites. Surprisingly, we found that U2AF65
does not bind to the polypyrimidine tract upstream of exon v5 directly. We provided
evidence that YB-1 activates splicing by facilitating the recruitment of U2AF65 to
weak polypyrimidine tracts through protein-protein interactions. In the mammalian
genome, the sequences at the 3’ splice site are highly degenerate. A fundamental
question about how such poor 3’ splice sites are recognized by the splicing machinery
has remained unclear. Our findings suggest a new model for the recognition of weak
3’ splice sites.
Caput epididymis-specific Cre transgenic mouse model and functions of miR-34a
in spermatogenesis
Shengsong Xie1, Juan Xu2, Jinxiong Han2, Xingxu Huang2*, Yonglian Zhang1,3*
1Shanghai Key Laboratory of Molecular Andrology, State Key Laboratory of
Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, People's
Republic of China
2Model Animal Research Center of Nanjing University, Nanjing, 210061, People's
Republic of China
3Shanghai Institute of Planned Parenthood Research, Shanghai, 200032, People's
Republic of China
*Correspondence
ylzhang@sibs.ac.cn (Yonglian Zhang) or xingxuhuang@mail.nju.edu.cn (Xingxu
Huang)
To study the function of genes in the caput epididymis using Cre/loxP system,
here, we reported that generated and characterized a constitutively active, principal
cells of middle/distal caput epididymis-specific, Cre-expressing transgenic mouse line,
which using the 1.8 kb fragment promoter of mouse Lcn5 gene. This transgenic
mouse expression of Cre beginning in the late stages of postnatal epididymis
development should be useful tool for future studies genes involved in the
post-testicular sperm maturation in epididymis.
Mammalian meiosis is unique to germ cells and a critical step in sexual
reproduction. Essential for these events are programmed DNA double strand breaks
(DSBs). By using bioinformatics method, miR-34a was predicted to target a number
of genes which involve in DSB repair pathway, indicating that its potential functions
in meiotic DSB. Using quantitative real time PCR assays, we identified that the
miR-34a was preferentially expressed in the mouse testis, and their levels were
up-regulated upon meiotic initiation during testicular development and in adult
spermatogenesis. By generating germ cell specific overexpression mice model, we
demonstrated that elevated miR-34a resulted in male infertility and meiotic arrest.
Meanwhile, the expression levels of target genes which directly regulate meiotic DSB
showed significant decline. Data will be presented to illustrate the gamatogenesis
failure caused by meiotic DSB repair pathway disorder.
Long noncoding RNAs with snoRNA ends
We describe the discovery of sno-lncRNAs, a class of nuclear-enriched
intron-derived long noncoding RNAs (lncRNAs) that are processed on both ends by
the snoRNA machinery. During exonucleolytic trimming, the sequences between the
snoRNAs are not degraded, leading to the accumulation of lncRNAs flanked by
snoRNA sequences but lacking 5’ caps and 3’ poly(A) tails. Such RNAs are widely
expressed in cells and tissues and can be produced by either box C/D or box H/ACA
snoRNAs. Importantly, the genomic region encoding one abundant class of
sno-lncRNAs (15q11-q13) is specifically deleted in Prader-Willi Syndrome (PWS).
The PWS region sno-lncRNAs do not colocalize with nucleoli or Cajal bodies, but
rather accumulate near their sites of synthesis. These sno-lncRNAs associate strongly
with Fox-family splicing regulators and alter patterns of splicing. These results thus
implicate a previously unannotated class of lncRNAs in the molecular pathogenesis of
PWS.
A Positive Role of Tet1 DNA Dioxygenase in the Regulation of AdultNeural
Progenitor Cell Proliferation
Abstract
DNA hydroxylation catalyzed by Tetdioxygenases occurs abundantly in
embryonic stem cells and neurons in mammals. However, its biological function in
vivo is largely unknown. Here we demonstrate that Tet1 plays an important role in
regulating neural progenitor cell proliferation in adult mouse brain. Mice lacking Tet1
exhibit impaired hippocampal neurogenesis associated with poor learning and
memory. The mutant mice also exhibit DNA hypermethylation and down-regulation
of genesrelated to the proliferation of neural progenitor cells in the adult hippocampus.
Our results indicate that Tet1 is positively involved in the epigenetic regulation of
neural progenitor cell proliferation in the adult brain.
microRNA serves as a mRNA surveillance system by eliminating nonsense
messages
Ya Zhao, Yao Zhang, Xue Zhang and Ligang Wu
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology,
Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, China
Abstract
Eukaryotic cells are constantly at risk of various mutations, among which nonsense mutations
are a severe type that will lead to pre-mature termination of translation, and result in the
expression of potentially harmful C-terminal truncated proteins. Accordingly, the cells have
evolved specialized mechanism, namely, nonsense mediated mRNA decay (NMD), to selectively
eliminate aberrant transcripts harboring pre-mature stop codons (PTCs). One such remarkable
mechanism is exon junction complex-mediated NMD (EJC-NMD), which depends on the
recognition of a downstream EJC by translating ribosomes that stalled at PTC during pioneer
round of translation. Although EJC-NMD is conserved among eukaryotes and can efficiently
reduce the abundance of many targeted mRNAs, certain restrictions such as dependence of EJC
and -50nt boundary rule limit its universility to all nonsense messages. MicroRNA (miRNA) is a
class of small, non-coding RNA that previously known to inhibit translation and/or promote RNA
degradation by imperfectly base-pairing with 3’UTR of target messages.
In this study, we
provide evidence that PTCs can potentiate miRNA mediated down-regulation of nonsense
messages by translocating miRNA responsive elements (miREs) from ORF to 3’UTR upon PTC
recognition. We show that APC (adenomatous polyposis coli), a tumor suppressor gene that is
naturally subject to high frequency of nonsense mutations within last exon, which escapes
EJC-NMD can nevertheless be repressed efficiently by miRNA induced NMD (miNMD).
Additional experiments show that miNMD differs from EJC-NMD by employing RISC complex
as effector proteins, substantial repression at translation level, as well as distinct boundary rule.
We further searched for the PTC mutation in the mRNA and analyzed its relative abundance of
HeLa cells and human samples by deep sequencing, and found additional messages that are
specifically subject to miNMD regulation rather than EJC-NMD. These findings indicate that in
addition to its role in tuning gene expression, miRNA may serve as an effective surveillance
system to recognize and eliminate mRNAs bearing PTCs.
Premature Termination Codons Are Recognized in the Nucleus
in A Reading-Frame Dependent Manner
Abstract
mRNAs containing premature termination codons (PTCs) are known to be
degraded via nonsense-mediated mRNA decay (NMD). Unexpectedly, we found
that mRNAs containing any type of PTC (UAA, UAG, UGA) are detained in the
nucleus whereas their wild-type counterparts are rapidly exported. This retention is
strictly reading-frame dependent.
Strikingly, our data indicate that translating
ribosomes in the nucleus proofread the frame and detect the PTCs in the nucleus.
Moreover, the shuttling NMD protein Upf1 specifically associates with PTC+ mRNA
in the nucleus and is required for nuclear retention of PTC+ mRNA. Together, our
data lead to a working model that PTCs are recognized in the nucleus by translating
ribosomes, resulting in recruitment of Upf1, which in turn functions in nuclear
retention of PTC+ mRNA. Nuclear PTC recognition adds a new layer of
proofreading for mRNA and may be vital for ensuring the extraordinary fidelity
required for protein production.