Genome-wide Characterization of cis-acting Elements in the Promoters of Key Carotenoid Pathway Genes from the Main Species of Genus Citrus

Carotenoids are indispensable for both human health and plant survival.Citrus,is one of the fruit crops richest in carotenoid compounds,with approximately 115 kinds of carotenoids;tremendous diversity in carotenoids composition and concentration exists among various species,showing different colors from nearly white to crimson.The carotenoid biosynthetic pathway and the key carotenogenic genes have been identified in citrus;however,the underlying regulatory mechanisms remain unclear.In this study,among the main species of genus Citrus(primitive,wild,and cultivated),we detected carotenoids in flavedo using High-Performance Liquid Chromatography,and analyzed variations in cis-acting elements in the promoters of key carotenoid pathway genes.Intriguingly,both carotenoid composition and content were generally increased during the evolution of citrus,and the corresponding variations in the promoters were identified,including the gain or loss of critical environmental stress-responsive elements and hormone-responsive elements,which are closely associated with carotenoid enhancement.In addition,pummelo has the most heat-responsive elements,but the Mangshan mandarin does not have this element in the promoters of PSY,which is highly related to their geographical origin and indicate that temperature is a critical environmental signal influencing carotenoid accumulation.Moreover,the abscisic acid-responsive motif was rich in almost all the seven species,but the ethylene-responsive motif was deficient,which demystified the unique phytohormone regulation mechanism of carotenoid accumulation in citrus.Overall,our study provides new insights into the molecular regulatory mechanism of carotenoid enhancement in the evolution of citrus,which can facilitate breeding and cultivation efforts to improve the nutritional quality and esthetic value in citrus and hopefully other fruit crops.

Genomic Analysis of MicroRNA Promoters and Their Cis-Acting Elements in Soybean

MicroRNAs （miRNAs） are derived from distinct loci in the genome and play crucial roles in RNA-mediated gene silencing mechanisms that regulate cellular processes during development and stress responses of plants. The miRNAs are approximately 21 nucleotides long and code for the complementary strand to a larger genic mRNA. They are often found within the complementary primary transcript （pri-miRNAs）. In the past few years, a growing number of soybean miRNAs have been discovered, however, little is known about the transcriptional regulation of these miRNAs. In this study, promoters and cis-acting elements of soybean miRNAs were analyzed using the genomic data for the first time. A total of 82 miRNAs were located among 122 loci in genome, some were present as double or multiple copies. Five clusters that included ten miRNAs were found in genome, and only one cluster share the same promoter. A total of 191 promoters from 122 loci of the soybean miRNA sequences were found and further analyzed. The results indicated that the conserved soybean miRNA genes had a greater proportion of promoters than that of non-conserved ones, and the distribution of the transcript start sites （TSSs） and TATA-boxes found had different motif styles between conserved and non-conserved miRNA genes. Furthermore, the cis-acting elements 5＇ of the TSSs were analyzed to obtain potential function and spatiotemporal expression pattern of miRNAs. The data obtained here may lead to the identification of specific sequences upstream of pre-miRNAs and the functional annotation of miRNAs in soybean.

MicroRNA Primary Transcripts and Promoter Elements Analysis in Soybean (Glycine max L. Merril.)

The importance of microRNA （miRNA） at the post-transcriptional regulation level has recently been recognized in both animals and plants. In recent years, many studies focused on miRNA target identification and functional analysis. However, little is known about the transcription and regulation of miRNAs themselves. In this study, the transcription start sites （TSSs） for 11 miRNA primary transcripts of soybean from 11 miRNA loci （of 50 loci tested） were cloned by a 5＂ rapid amplification of cDNA ends （5＂ RACE） procedure using total RNA from 30-d-old seedlings. The features consistent with a RNA polymerase II mechanism of transcription were found among these miRNA loci. A position weight matrix algorithm was used to identify conserved motifs in miRNA core promoter regions. A canonical TATA box motif was identified upstream of the major start site at 8 （76%） of the mapped miRNA loci. Several cis-acting elements were predicted in the 2 kb 5＂ to the TSSs. Potential spatial and temporal expression patterns of the miRNAs were found. The target genes for these miRNAs were also predicted and further elucidated for the potential function of the miRNAs. This research provides a molecular basis to explore regulatory mechanisms of miRNA expression, and a way to understand miRNA-mediated regulatory pathways and networks in soybean.

Analysis on Sequence and Elements of Cucumisin Promoter

Abstract Leaves of melon were collected and extracted by the CTAB method for total DNA which was used for PCR amplification, obtaining the gene sequence of cucumisin promoter. The sequence results were processed and analyzed with DNAman, DNAstar and other softwares, and bioinformatic element analysis was performed with PlantCARE and PLACE. The analysis results showed that the cucumisin promoter shared 100%, 99% and 99% homology with AY055805, LN713264 and LN681897, respectively. The promoter sequence contains a variety of c/s-acting elements common in fruit promoters of higher plants such as TATA-Box and CAAT-Box, and light-responsive elements, some of which involved in ABA and VP1 responsiveness and salicylic acid responsiveness. This study provides a scien- tific basis for further research on genetic engineering of fruits.

Cis-acting regulatory elements： from random screening to quantitative design

The cis-acting regulatory elements, e.g., promoters and ribosome binding sites （RBSs） with various desired properties, are building blocks widely used in synthetic biology for fine tuning gene expression. In the last decade, acquisition of a controllable regulatory element from a random library has been established and applied to control the protein expression and metabolic flux in different chassis cells. However, more rational strategies are still urgently needed to improve the efficiency and reduce the laborious screening and multifaceted characterizations. Building precise computational models that can predict the activity of regulatory elements and quantitatively design elements with desired strength have been demonstrated tremendous potentiality. Here, recent progress on construction of cis- acting regulatory element library and the quantitative predicting models for design of such elements are reviewed and discussed in detail.

Identification of the 2-tridecanone cis-acting element in the promoter of cytochrome P450 CYP6B7 in Helicoverpa armigera

The expression level of cytochrome P450 genes in insects can be induced by plant allelochemicals,which is important for insects to adapt to host plants.Cytochrome P450CYP6B 7has been reported to be involved in pyethroid insecticide resistance in Heli- coverpa armigera,and its transcription level was induced by some inducers.Currently,the regulatory mechanism of the induced expression of CYP6B7remains unknown,although it is very important for understanding the detoxification mechanism to allelochemicals in host plants.The objective of the present study was to investigate the eis-acting ele- ment in the promoter of CYP6B7 mediating the inducible up-regulation of CYP6B7in H.armigera by 2-tridecanone.The promoter region of CYP6B7was cloned by genome walking technique and analyzed by transient transfeetion assay.Progressive 5'deletion of the promoter region of CYP6B7revealed that the relative luciferase activity of construct -320/+232could be significantly induced by 2-trideeanone.Further stepwise deletion between -320 and -238 bp found that construct -292/+232 could also be significantly induced by 2-tridecanone,but the adjacent construct -256/+232could not,suggesting the essential role of the sequence between -292 and --257 bp for 2-tridecanone induction. Nucleotide mutations between -292 and -281 bp had no influence on the induction ef- fect by 2-tridecanone,but nucleotide mutations between -280 and -257 bp significantly decreased the induction effect.These results demonstrated that the cis-acting element for 2-trideeanone induction was between -280 and -257 bp in the promoter of CYP6B7.

Synthetic promoters consisting of defined cis-acting elements link multiple signaling pathways to probenazole-inducible system

Probenazole （3-allyloxy-l,2-benzisothiazole-1,1-dioxide, PBZ）, the active component of Oryzemate, could induce systemic acquired resistance （SAR） in plants through the induction of salicylic acid （SA） biosynthesis. As a widely used chemical inducer, PBZ is a good prospect for establishing a new chemical-inducible system. We first designed artificially synthetic promoters with tandem copies of a single type of cis-element （SARE, JERE, GCC, GST1, HSRE, and W-box） that could mediate the expression of the tS-glucuronidase （GUS） reporter gene in plants upon PBZ treatment. Then we combined different types of elements in order to improve inducibility in the PBZ-inducible system. On the other hand, we were surprised to find that the cis-elements, which are responsive to jasmonic acid （JA） and ethylene, also responded to PBZ, implying that SA, JA, and ethylene pathways also would play important roles in PBZ＇s action. Further analysis demonstrated that PBZ also induced early events of innate immunity via a signaling pathway in which Ca2＋ influx and mitogen-activated protein kinase （MAPK） activity were involved. We constructed synthesized artificial promoters to establish a PBZ chemical-inducible system, and preliminarily explored SA, JA, ethylene, calcium, and MAPK signaling pathways via PBZ-inducible system, which could provide an insight for in-depth study.

Interactions between the nuclear matrix proteins and the 5'-flanking cis-acting elements of the human ε-globin gene

An erythroid-specific nuclear matrix protein (termed ε-NMP_k) in K562 cells, which can specifically bind to the positive stage-specific regulatory element (ε-PRE Ⅱ, -446—-419 bp) upstream of the human ε-globin gene, has been identified by using gel mobility shift assay.Meanwhile, Southwestern blotting assay showed that the nuclear matrix protein ε-NMP_k in K562,cells may be composed of two polypeptides ( ～ 40 ku). In addition, it is observed in the gel mobility shift assay that the nuclear matrix proteins from K562, HEL and Raji cells can bind to the silencer DNA ( - 392— -177 bp) in the 5’-flanking sequence of human ε-globin gene respectively. However, the shift band K detected in K562 cells is different from shift band H/R in HEL and Raji cells, suggesting that a common nuclear matrix protein may exist in HEL and Raji cells. Results show that the nuclear matrix protein may play an important role in the regulation of the human ε-globin gene expression.

Three 5’-flanking Regions of crtO Encoding β-carotene Oxygenase in Haematococcus pluvialis

Three separate 5＇-flanking regions （1.1 kb, 1.9 kb and 2.2 kb） of crtO were cloned through walking upstream. Results of sequence analysis show that three 5＇-flanking regions of crtO might have some similar putative cis-acting elements such as ABA （abscisic acid）-responsive element （ABRE）, C-repeat/dehydration responsive element （C-repeat/DRE）, light-responsive element （G-box, GAG-motif, I-box and ATC-motif）, wound-responsive element （WUN-motif）, auxin-responsive element （TGA-element）, MeJA-responsive element （TGACG-element） and MYB binding site （MBS）, except for typical TATA box or CCAAT box. These findings might mean diversiform regulatory patterns of crtO being in astaxanthin biosynthesis of Haematococcus pluvialis.

Regulatory Analysis of IPP Isomerase Gene in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvia/is uniquely accumulates carotenoids in the cytoplasm and in late developmental stages turns deep-red in color because of accumulation of astaxanthin in the cytosol. The enzyme, isopentenyl pyrophosphate （IPP） isomerase, plays a key role in astaxanthin biosynthesis of H. pluvialis. In this paper, two separate 5＇-flanking regions （1.8 kb and 2.5 kb） of IPP isomerase gene was cloned through walking upstream firstly. Results of sequence analysis =showed that two separate 5＇-flanking regions of IPP isomerase gene might have similar putative cis-acting elements such as ABA （abscisic acid）-responsive element （ABRE）, drought-responsive element （DRE/C-repeat）, light-responsive element （G-box, GAG-motif, I-box and ATC-motif）, heat-shock element （HSE）, wound-responsive element （WUN-motif）, SA （salicylic acid）-responsive element （TCA-element）, auxin-responsive element （TGA-element）, MeJA （methyl jasmonate）-responsive element （TGACG-element）, enhancer-like element involved in anoxic specific inducibility （GC-motif） and MYB binding sites （MBS and MRE）, except for typical TATA box or CCAAT box, which exhibit diversiform transcriptional patterns of IPP isomerase gene in astaxanthin biosynthesis of Haematococcus pluvialis.

Isolation and analysis of a TIR-specific promoter from poplar

A 5＇ flanking region of the well-conserved Toll/interleukin-1 receptor domain （TIR）-encoding sequence was isolated from the genomic DNA ofMelampsora magnusiana Wagner resistant clones of hybrid triploid poplars [（Populus tomentosa × P. bolleana） × P. tomentosa]. Sequencing results and alignment analysis show that the obtained TIR-specific promoter （named as PtTIRp01） was 1,732 bp in length; moreover 3＇ region of the PtTIRp01 contains a responds to the 5＇ composition of TIR-NBS type gene PtDRG02, of 747 bp long 5＇ region of TIR-NBS type gene PtDRG02 and its 398 bp complete TIR-encoding sequence, which significantly corindicating that the obtained TIR-specific promoter region consists upstream region of promoter （985 bp）. It was found that the 5＇ region of TIR-NBS type gene PtDRG02 was characterized in the downstream region of the transcriptional start, named as 5＇-untranslated region （5＇ UTR）, consisting of one 93 bp 5＇-untranslation exon, one 213 bp intron and one 441 bp TIR-encoding open reading frame （ORF）. In addition, several putative cis-acting motifs were present in the obtained TIR-specific promoter of PtDRG02, including one TATA box, one GC-rich, one AT-rich, one P-box, one 3-AF1 binding site, two CAAT boxes, two GT-1 motifs, three typical W-boxes, four I-boxes, and one multi-cis-acting fragment （MCF）. The latter contains five types of regulatory elements （E4, G-box, ABRE motif, box 1 and HVA 1 s）, most of which were homologous to the cis-acting regulatory elements involved in the activation of defense genes in plants. Thus, it can be suggested that TIR-specific promoter might be a pathogen-inducible promoter and be necessary for the inducible expression of defense-related genes. Key words Toll/interleukin- 1 receptor domain, promoter, Cis-acting element, poplar

Functional analysis of cis-acting sequences regulating root-specific expression in transgenic tobacco

Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-specific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expression were studied by fusing them to the β-glucuronidase (GUS) report gene with different directions. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The data suggested that the cis-acting element of TobRB7 gene direct GUS expression not only as root-specific but also as bidirectional. In our studies, the short fragment, RSF2, performed stronger activity than RSF1 with any direction. The stronger activity of GUS expression was determined by reverse inserting of RSF1 or RSF2 than positive inserting.

Harpin_(xoo) and Its Functional Domains Activate Pathogen-inducible Plant Promoters in Arabidopsis

Harpins are bacterial proteins that can enhance plant growth and defense against pathogens and insects. To elaborate whether harpins perform the diverse functions in coordination with the activation of specific promoters that contain particular elements, we cloned pathogen-inducible plant promoters PPP1, PPP2, and PPP3 from tobacco and investigated their responses to harpinxoo or its truncated fragments DEG, DIR, and DPR (domains for enhancing plant growth, insect resistance and pathogen resistance). PPP1 contains an internal repeat composed of two tandem 111bp fragments; 111bp in the repeat was deleted in PPP2. PPP3 contains a bacteria-inducible element; PPP1 and PPP2 additionally contain TAC-1 and Eli boxes inducible correspondingly by salicylic acid (SA) and elicitors. Function of cloned PPPs was confirmed based on their activation in transgenic Arabidopsis plants by Ralstonia solanacearum (Ralston) or SA. Harpinxoo, DEG, DIR, or DPR activated PPP1 and PPP2 but not PPP3, consistent with the presence of Eli boxes in promoters. PPP1 was ca. 3-fold more active than PPP2, suggesting that the internal repeat affects levels of the promoter activation.

In silico Analysis of osr40c1 Promoter Sequence Isolated from Indica Variety Pokkali

The promoter region of a drought and abscisic acid （ABA） inducible gene, osr40c1, was isolated from a salt-tolerant indica rice variety Pokkali, which is 670 bp upstream of the putative translation start codon. In silico promoter analysis of resulted sequence showed that at least 15 types of putative motifs were distributed within the sequence, including two types of common promoter elements, TATA and CAAT boxes. Additionally, several putative cis-acing regulatory elements which may be involved in regulation of osr40c1 expression under different conditions were found in the 5′-upstream region of osr40c1. These are ABA-responsive element, light-responsive elements （ATCT-motif, Box I, G-box, GT1-motif, Gap-box and Sp1）, myeloblastosis oncogene response element （CCAAT-box）, auxin responsive element （TGA-element）, gibberellin-responsive element （GARE-motif） and fungal-elicitor responsive elements （Box E and Box-W1）. A putative regulatory element, required for endosperm-specific pattern of gene expression designated as Skn-1 motif, was also detected in the Pokkali osr40c1 promoter region. In conclusion, the bioinformatic analysis of osr40c1 promoter region isolated from indica rice variety Pokkali led to the identification of several important stress-responsive cis-acting regulatory elements, and therefore, the isolated promoter sequence could be employed in rice genetic transformation to mediate expression of abiotic stress induced genes.

Genome-wide identification and in silico gene expression analysis of the related to ABI3/VP1(RAV)transcription factor family in barley(Hordeum vulgare L.)

RAV(Related to ABI3/VP1)transcription factors are unique members of the AP2-ERF superfamily with AP2 and B3 domains and play important roles in the regulation of seed germination,plant growth,and stress response.In the study,7 RAV genes,named HvRAVs,were identified in barley based on the available genome sequences.While five of the seven HvRAVs were located on chromosome 3,HvRAV5 and HvRAV7 were located on chromosome 1 and 4,respectively.Six of the predicted HvRAVs were intron-less,except HvRAV2,which had one intron.HvRAV proteins have shown basic,instable,and hydrophilic properties.The AP2 domain specific RAYD and WLG motifs were detected in all HvRAV proteins.Besides,B3 repression domain,R/KLFGV,is also found in the C-terminal of HvRAVs.HvRAVs were found to have stress-related cis-acting elements,including MYB,MYC,and W-BOX.HvRAV2 was predicted to have no GARE motifs,TATCCCA or TAACAA(G/A),and LTREs.Under drought conditions,the expression level of HvRAVs did not significantly change in a drought-sensitive barley genotype,whereas HvRAV5 and HvRAV7 were dramatically down-regulated in a drought-tolerant genotype.Expression of HvRAV5 was also inhibited by salinity.HvRAV7 was strongly induced by plant pathogen attack.Only HvRAV6 was induced by exogenous gibberellin application and during the germination process.Interestingly,HvRAV6 transcript was detected higher than other HvRAVs in all stress and control conditions as well as during germination.In silico analyses have shown that HvRAVs play a role in response to different abiotic and biotic stresses as well as in plant development.However,the extensive biological roles of HvRAV genes in plant development and in response to abiotic and biotic stresses need further investigation.

Genome-wide identification and characterization of the abiotic-stress-responsive lipoxygenase gene family in diploid woodland strawberry(Fragaria vesca)

Lipoxygenase(LOXs)is a kind of dioxygenase without heme and iron,which plays an important role in the development and adaptation of many plants to the environment.However,the study of strawberry LOX gene family has not been reported.In this study,14 LOX genes were identified from the diploid woodland strawberry genome.The phylogenetic tree divides the FvLOX gene into two subfamilies:9-LOX and 13-LOX.Gene duplication event analysis showed that whole-genome duplication(WGD)/segmental duplication and dispersed duplication effectively promoted the expansion of strawberry LOX family.QRT-PCR analysis showed that FvLOX genes were expressed in different tissues.Expression profile analysis showed that FvLOX1 and FvLOX8 were up-regulated under low temperature stress,FvLOX3 and FvLOX7 were up-regulated under drought stress,FvLOX6 and FvLOX9 were up-regulated under salt stress,FvLOX2,FvLOX3 and FvLOX6 were up-regulated under salicylic acid(SA)treatment,FvLOX3,FvLOX11 and FvLOX14 were up-regulated under methyl jasmonate(MeJA)treatment,FvLOX4 and FvLOX14 were up-regulated under abscisic acid(ABA)treatment.Promoter analysis showed that FvLOX genes were involved in plant growth and development and stress response.We analyzed and identified the whole genome of strawberry FvLOX family and characterized a variety of FvLOX candidate genes involved in abiotic stress response.This study laid a theoretical and empirical foundation for the response mechanism of strawberry to abiotic stress.

Cloning and Analysis of the Promoters of Two OsRhoGDIs Genes from Rice

OsRacD, belonging to rice （Oryza sativa L. ssp. japonica） Rho family of the small GTPases, is a pivotal gene involved in rice photoperiod fertility conversion of photoperiod sensitive genic male sterile rice which influences the rice fertility via controlling the pollen tube growth. Using OsRacD as bait, two GDP dissociation inhibitor genes designated as OsRhoGDI1 and OsRhoGDI2 were screened by yeast two-hybrid system. To further study the regulation mechanism of OsRhoGDIs, and also the relationships between OsRhoGDIs and OsRacD, the upstream regulation sequences of OsRhoGDI1 and OsRhoGDI2 were cloned by PCR, and the cis-elements in the promoters were predicted using internet tools in this study. The results showed that there were several kinds of response elements, such as phytohormone response elements, lightregulated elements and adversity induced elements in the promoters of OsRhoGDIs, some of which were similar. Comparing with the promoter of OsRacD, several pollen tube germination related elements were found. On one hand, the expression and regulation mechanism of OsRhoGDIs were complex, they may be regulated by several signaling pathways commonly, and the regulation mechanisms were similar to some extent. On the other hand, it was suggested that the fertility of photoperiod sensitive genic male sterile rice may be controlled by the cooperative expression of OsRhoGDIs and OsRacD.

A novel cold-inducible promoter,PThCAP from Tamarix hispida,confers cold tolerance in transgenic Arabidopsis thaliana

Our previous studies have revealed that the Th CAP gene plays a vital role in transgenic Populus(P.davidiana 9 P.bolleana) in response to cold stress.However,the regulatory mechanism of Th CAP gene expression has been unclear.In this study,the 50 flanking region of the Th CAP promoter(PTh CAP) was cloned using a genomewalking method.By analyzing cis-acting regulatory elements of PTh CAP,a DRE motif and MYC and MYB elements were found to be located in the promoter.To identify the regulatory elements that control the expression of the Th CAP gene promoter,a series of deletion derivatives ofPTh CAP,P1–P5,from the translation start code(-1538,-1190,-900,-718 and-375 bp),were fused to the GUS reporter gene,and then each deletion was stably introduced into Arabidopsis thaliana plants.Deletion analysis of the promoter suggested that only the P2 fragment had strong GUS expression in leaves and roots of A.thaliana exposed to low temperature stress.These results suggest that this290-bp region(-1190 to-900 bp),as an important part in PTh CAP,was associated with cold tolerance of A.thaliana.Our results provide evidence for the regulatory mechanism of Th CAP gene involved in the response to cold stress,and that the gene is promising candidate gene for genetic improvement of crops.

Grain proteomic analysis reveals central stress responsive proteins caused by wheat-Haynaldia villosa 6VS/6AL translocation

Haynaldia villosa(2n=14,VV),a wild grass of the subtribe Triticeae,serves as potential gene resources for wheat genetic improvement.In this study,the proteome characterization during grain development of Yangmai 5 and Yangmai 5-H.villosa 6VS/6AL translocation line was investigated by a comparative proteomic approach.Two-dimensional electrophoresis identified 81 differentially accumulated proteins(DAPs)during five grain developmental stages in wheat-H.villosa translocation line.These proteins were mainly involved in stress defense,storage protein,energy metabolism,protein metabolism and folding,carbon metabolism,nitrogen metabolism,and starch metabolism.In particular,6VS/6AL translocation led to significant upregulation of 36 DAPs and specific expression of 11 DAPs such as chitinase,thaumatin-like proteins,glutathione transferase,α-amylase inhibitor,heat shock proteins,and betaine aldehyde dehydrogenase.These proteins mainly involved in biotic and abiotic stress responses.Further analysis found that the upstream 1500 promoter regions of these stress-responsive DAP genes contained multiple high-frequency cis-acting elements related to stress defense such as abscisic acid response element ABRE,methyl jasmonate(MeJA)-response element TGACG-motif and CGTCA-motif involved in oxidative stress and antioxidant response element(ARE).RNA-seq and RT-qPCR analyses revealed the high expression of these stress-defensive DAP genes in the developing grains,particularly at the early-middle grain filling stages.Our results demonstrated that 6VS chromosome of H.villosa contains abundant stress-defensive proteins that have potential values for wheat genetic improvement.

Structure-function relationship in viral RNA genomes: The case of hepatitis C virus

The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus(HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5'-untranslatable regions(5'UTRs) and 3'UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5' terminus of the viral genome and regulated by distal functional RNA domains placed at the 3' end. Subsequent RNA replication strongly depends on the 3'UTR folding and is also influenced by the 5' end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNARNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.