RNA polymerase IV

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RNA polymerase IV (or RNAP IV) is an enzyme that synthesizes small interfering RNA (siRNA) in plants, which silence gene expression. [1] [2] [3] RNAP IV belongs to a family of enzymes that catalyze the process of transcription known as RNA Polymerases, which synthesize RNA from DNA templates. [4] Discovered via phylogenetic studies of land plants, genes of RNAP IV are thought to have resulted from multistep evolution processes that occurred in RNA Polymerase II phylogenies. [5] Such an evolutionary pathway is supported by the fact that RNAP IV is composed of 12 protein subunits that are either similar or identical to RNA polymerase II, and is specific to plant genomes. [6] Via its synthesis of siRNA, RNAP IV is involved in regulation of heterochromatin formation in a process known as RNA directed DNA Methylation (RdDM). [1] [2]

Contents

Discovery

Phylogenetic Studies

Structure of RNA Polymerase II as a model for the structure of RNA Polymerase IV, which is currently under research. RNA Polymerase II.png
Structure of RNA Polymerase II as a model for the structure of RNA Polymerase IV, which is currently under research.

Phylogenetic studies of land plants have led to the discovery of RNA Polymerase IV. [5] Analysis of the largest (RPD1) and second-largest subunits (RPD2) of RNAP IV were analogous to the Blast searches of RNAP II genes. [5] Genes for RPD1 and RPD2 were found in all terrestrial plants, and the largest gene was found in the algal taxon, Charale. Further analysis of the origin of the protein indicates a gene duplication event of the largest subunit which suggested that the duplication event occurred after the divergence of Charales and land plants and algae. [5] Specifically, the largest subunit in RNAP II formed RPD1 through a duplication event and the RPD2 gene arose due to a divergence. Evidence of these duplication events imply that the RNAP IV genes come from RNAP II phylogenies in a multistep process. In other words, the divergence of the first subunit is the first step of multiple in the evolution of new RNAPs. [5] RNAP IV also shares multiple subunits with RNAP II, in addition to the largest and second largest subunits, which was also suggested by continuous duplication events of particular lineages. [7]

Distinction Between RNAP IV and RNAP V

Arabidopsis expresses two forms of RNAP IV, formerly referred to as RNAP IVa and RNAP IVb, which differ at the largest subunit and have non redundant actions. [8] Efficient silencing of transposons requires both RNAP IV forms while only RNAP IVa is required for basal silencing. This finding suggested the requirement of both forms for the mechanism of transposon methylation. [8] Later experiments have shown that what was once thought to be two forms of RNAP IV are actually two structurally and functionally distinct polymerases. [9] RNAP IVa was specified to be RNAP IV while RNAP IVb became known as RNAP V. [9]

Structure

RNA Polymerase IV is composed of 12 protein subunits that are either similar or identical to the 12 subunits composing RNA Polymerase II. Only four subunits distinguish RNAP IV structure from RNAP II and RNAP V. RNA Polymerase V differs from RNAP II by six subunits, indicating that both RNAP IV and RNAP V evolved from RNAP II in plants. [6] In Arabidopsis, two unique genes were found to encode subunits that distinguish RNAP IV from RNAP II. [10] The largest subunit is encoded by NRPD1 (formerly NRPD1a), while the second largest subunit is encoded by NRPD2 and is shared with RNAP V. [1] These subunits contain carboxyl-terminal domains (CTDs) which are necessary for the production of 20-30% of the siRNAs produced by RNA Polymerase IV, yet are not required for DNA methylation. [11]

Function

Model representing the stepwise process of RNA Directed DNA Methylation via RNA Polymerase IV. RNA Pol IV Mechanism.png
Model representing the stepwise process of RNA Directed DNA Methylation via RNA Polymerase IV.

There is evidence that RNA Polymerase IV (RNAP IV) is responsible for producing heterochromatin, as dysfunction of either RNAP IV catalytic subunit (NRPD1 and NRPD2) disrupts the formation of heterochromatin. [2] As heterochromatin is the silenced portion of DNA, it is formed when RNAP IV amplifies production of small interfering RNAs (siRNA) that are responsible for methylating cytosine bases in DNA; this methylation silences segments of the genetic code, which can still be transcribed into mRNA but not translated into proteins. [3] [12] RNAP IV is involved in setting the methylation patterns in the 5S genes during plant maturation, resulting in the development of adult features in plants. [13]

Mechanism

In the first step of heterochromatin formation, RNAP IV couples with an RNA-dependent RNA polymerase known as RDR2 to make a double stranded precursor to siRNA. [14] Next, DICER-Like Protein 3 (DLP3), an enzyme which slices double stranded RNA substrates, cleaves the double stranded precursor into siRNAs that are each 24 nucleotides long. [15] These siRNAs are then methylated at their 3’ ends by a protein known as HUA ENHANCER 1 (HEN1). [16] Finally, these methylated siRNAs complex with a protein known as ARGONAUTE-4 (AGO4) in order to form the silencing complex that can perform the required methylation for heterochromatin production. [17] This process is referred to as RNA-directed DNA Methylation (RdDM) or Pol IV-mediated silencing as the introduction of these methyl groups by siRNAs silence both transposons and repetitive sequences of DNA. [1]

Regulation

SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1) is a protein that interacts with RNAP IV and is critical in its regulation through methylation. SHH1 can only bind to chromatin at specified “marked” segments, as its “SAWADEE” domain is a chromatin binding domain that probes for unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail of chromatin; its binding pockets then attach to chromatin at these sites and allow RNAP IV occupancy at these same loci. In this manner, SHH1 functions to enable RNAP IV recruitment and stability at the most actively targeted genomic loci in RdDM in order to promote the previously mentioned siRNA biogenesis of 24 nucleotide-long siRNA. Furthermore, it binds to repressive histone modifications, and any mutations that interfere with this process are associated with a reduction in DNA methylation and siRNA production. [18] Regulation of siRNA production by RNAP IV through this mechanism results in major downstream effects, as the siRNAs produced in this manner defend the genome against the proliferation of invading viruses and endogenous transposable elements. [19]

Related Research Articles

Histone Family proteins package and order the DNA into structural units called nucleosomes.

In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are wrapped into 30-nanometer fibers that form tightly packed chromatin. Histones prevent DNA from becoming tangled and protect it from DNA damage. In addition, histones play important roles in gene regulation and DNA replication. Without histones, unwound DNA in chromosomes would be very long. For example, each human cell has about 1.8 meters of DNA if completely stretched out, however when wound about histones, this length is reduced to about 90 micrometers (0.09 mm) of 30 nm diameter chromatin fibers.

Heterochromatin is a tightly packed form of DNA or condensed DNA, which comes in multiple varieties. These varieties lie on a continuum between the two extremes of constitutive heterochromatin and facultative heterochromatin. Both play a role in the expression of genes. Because it is tightly packed, it was thought to be inaccessible to polymerases and therefore not transcribed, however according to Volpe et al. (2002), and many other papers since, much of this DNA is in fact transcribed, but it is continuously turned over via RNA-induced transcriptional silencing (RITS). Recent studies with electron microscopy and OsO4 staining reveal that the dense packing is not due to the chromatin.

Transcription (biology) Process of copying a segment of DNA into RNA

Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called non-coding RNAs (ncRNAs). Averaged over multiple cell types in a given tissue, the quantity of mRNA is more than 10 times the quantity of ncRNA. The general preponderance of mRNA in cells is valid even though less than 2% of the human genome can be transcribed into mRNA, while at least 80% of mammalian genomic DNA can be actively transcribed, with the majority of this 80% considered to be ncRNA.

RNA polymerase

In molecular biology, RNA polymerase, is an enzyme that synthesizes RNA from a DNA template.

In molecular biology and genetics, transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing the mRNA that encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in multicellular eukaryotes, as studied in evolutionary developmental biology.

Transcription preinitiation complex Complex of proteins necessary for gene transcription in eukaryotes and archaea

The preinitiation complex is a complex of approximately 100 proteins that is necessary for the transcription of protein-coding genes in eukaryotes and archaea. The preinitiation complex positions RNA polymerase II at gene transcription start sites, denatures the DNA, and positions the DNA in the RNA polymerase II active site for transcription.

Repressor

In molecular genetics, a repressor is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA. An RNA-binding repressor binds to the mRNA and prevents translation of the mRNA into protein. This blocking or reducing of expression is called repression.

RNA polymerase II Protein complex that transcribes DNA

RNA polymerase II is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNAP enzymes found in the nucleus of eukaryotic cells. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase. A wide range of transcription factors are required for it to bind to upstream gene promoters and begin transcription.

General transcription factor

General transcription factors (GTFs), also known as basal transcriptional factors, are a class of protein transcription factors that bind to specific sites (promoter) on DNA to activate transcription of genetic information from DNA to messenger RNA. GTFs, RNA polymerase, and the mediator constitute the basic transcriptional apparatus that first bind to the promoter, then start transcription. GTFs are also intimately involved in the process of gene regulation, and most are required for life.

The family of heterochromatin protein 1 (HP1) consists of highly conserved proteins, which have important functions in the cell nucleus. These functions include gene repression by heterochromatin formation, transcriptional activation, regulation of binding of cohesion complexes to centromeres, sequestration of genes to nuclear periphery, transcriptional arrest, maintenance of heterochromatin integrity, gene repression at the single nucleosome level, gene repression by heterochromatization of euchromatin and DNA repair. HP1 proteins are fundamental units of heterochromatin packaging that are enriched at the centromeres and telomeres of nearly all Eukaryotic chromosomes with the notable exception of budding yeast, in which a yeast-specific silencing complex of SIR proteins serve a similar function. Members of the HP1 family are characterized by an N-terminal chromodomain and a C-terminal chromoshadow domain, separated by a Hinge region. HP1 is also found at euchromatic sites, where its binding correlates with gene repression. HP1 was originally discovered by Tharappel C James and Sarah Elgin in 1986 as a factor in the phenomenon known as position effect variegation in Drosophila melanogaster.

RNA-induced transcriptional silencing (RITS) is a form of RNA interference by which short RNA molecules – such as small interfering RNA (siRNA) – trigger the downregulation of transcription of a particular gene or genomic region. This is usually accomplished by posttranslational modification of histone tails which target the genomic region for heterochromatin formation. The protein complex that binds to siRNAs and interacts with the methylated lysine 9 residue of histones H3 is the RITS complex.

RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA (dsRNA). RNA silencing mechanisms are highly conserved in most eukaryotes. The most common and well-studied example is RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA) and its subspecies repeat associated small interfering RNA (rasiRNA).

Eukaryotic transcription

Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes comes in three variations, each translating a different type of gene. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.

FACT is a heterodimeric protein complex that affects eukaryotic RNA polymerase II transcription elongation both in vitro and in vivo. It was discovered in 1998 as a factor purified from human cells that was essential for productive, in vitro Pol II transcription on a chromatinized DNA template.

RNA polymerase II holoenzyme is a form of eukaryotic RNA polymerase II that is recruited to the promoters of protein-coding genes in living cells. It consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins.

Transposable elements are short strands of repetitive DNA that can self-replicate and translocate within the eukaryotic genome, and are generally perceived as parasitic in nature. Their transcription can lead to the production of dsRNAs, which resemble retroviruses transcripts. While most host cellular RNA has a singular, unpaired sense strand, dsRNA possesses sense and anti-sense transcripts paired together, and this difference in structure allows an host organism to detect dsRNA production, and thereby the presence of transposons. Plants lack distinct divisions between somatic cells and reproductive cells, and also have, generally, larger genomes than animals, making them an intriguing case-study kingdom to be used in attempting to better understand the epigenetics function of transposable elements.

RNA polymerase V is a multisubunit plant specific RNA polymerase found in nucleus. Together with RNA polymerase IV required for normal function and biogenesis of small interfering RNA (siRNA). Pol V is involved in siRNA-directed DNA methylation pathway which leads to heterochromatic silencing.

DDM1, Decreased DNA Methylation I, is a plant gene that encodes a nucleosome remodeler which facilitates DNA methylation. The DDM1 gene has been described extensively in Arabidopsis thaliana and also in maize. The protein has been described to be similar to the SWI2/SNF2 chromatin remodeling proteins.

Julie Law American molecular and cellular biologist.

Julie Law is an American molecular and cellular biologist. Law's pioneering work on DNA methylation patterns led to the discovery of the role of the CLASSY protein family in DNA methylation. Law is currently an associate professor at the Salk Institute for Biological Studies.

RNA-directed DNA methylation RNA-based gene silencing proces

RNA-directed DNA methylation (RdDM) is a biological process in which non-coding RNA molecules direct the addition of DNA methylation to specific DNA sequences. The RdDM pathway is unique to plants, although other mechanisms of RNA-directed chromatin modification have also been described in fungi and animals. To date, the RdDM pathway is best characterized within angiosperms, and particularly within the model plant Arabidopsis thaliana. However, conserved RdDM pathway components and associated small RNAs (sRNAs) have also been found in other groups of plants, such as gymnosperms and ferns. The RdDM pathway closely resembles other sRNA pathways, particularly the highly conserved RNAi pathway found in fungi, plants, and animals. Both the RdDM and RNAi pathways produce sRNAs and involve conserved Argonaute, Dicer and RNA-dependent RNA polymerase proteins.

References

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