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Archive for the ‘Gene Expression’ Category

RNAi is fast emerging as a wonderful tool for inhibiting gene expression in a sequence specific manner. The applications of this technology are two-fold: to study gene function, and use as a therapeutic agent in treating many diseases. As a therapeutic agent it finds applications in antiviral treatments because RNAi has been shown to successfully inhibit virus replication. In a report published on 8 November, 2007, in the Retrovirology, Naito et al., describe the design of antiviral siRNA targeted against HIV-I. To create effective antiviral siRNAs against HIV is a daunting task as the virus mutates at a very high frequency. The researchers first analyzed the HIV-I group M sequences available in the Los Alamos HIV Sequence Database and then found those regions which are highly conserved. Using these conserved regions as target sites, they designed optimal antiviral siRNAs. 21-mer siRNA sequences were generated for all the possible HIV-I sequences. The conserved sequences identified in HIV-I genome included the TATA sequence, polyadenylation signal (AAUAAA), regions essential for viral replication regulation, the primer activation signal (PAS), primer binding site (PBS), packaging signal (ψ), central polypurine tract (cPPT), central termination sequence (CTS), and 3 polypurine tract (3 PPT). A total of 216 highly conserved (>70%) siRNA targets were identified. 41 siRNAs (23 siRNAs out of 216 mentioned above and 18 more siRNAs targeted against moderately conserved regions) were subjected to target mRNA cleavage assay for functional validation using real-time RT-PCR. HeLa cells were cotransfected with vector expressing reporter mRNA that contains the siRNA target site and the corresponding siRNA. Then the potency of siRNAs was monitored by real-time RT-PCR. siRNAs were evaluated for their antiviral efficacy against HIV subtypes B, B’, C and CRF01_AE.
The study clearly demonstrated that 39 out of the 41 siRNAs gave more than >60% silencing; and 26 of the 41 siRNAs effectively inhibited viral replication of all four strains by >80%. The results of the study are quite remarkable and point towards the efficient use of siRNA for inhibiting viral gene expression and replication. The study also paved the way for using siRNAs against divergent HIV-I strains. However, the extreme genetic diversity and high mutation rate of the virus has hindered the creation of a single siRNA effective against all HIV-I strains found in the world. The use of this technology for as a highly effective treatment might be possible in the future and this study could be applied to other pathogens like SARS, influenza virus, etc.

Read the article here.


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piRNAs

Piwi-interacting RNAs (piRNA) are a class small of RNA molecules that are expressed uniquely in mammalian spermatogenic cell lines. These are 26–31 nucleotides long and bigger that miRNAs and siRNAs. They are so named because of their capability of forming RNA-protein complexes with Piwi proteins. Piwi proteins are part of the family of Argonaute proteins, which are defined by the PAZ (Piwi Argonaut and Zwille) domain and the PIWI domain. Argonaute proteins interact with small RNAs through PAZ and PIWI domains. A small RNA guides the Argonaute protein to its target molecule, which leads to gene silencing. MIWI, MIWI2 and MILI, three Piwi subfamily proteins, are essential for spermatogenesis in mice. piRNAs are involved in RNA silencing via the formation of RISC. The biogenesis pathway of piRNAs has not been clearly elucidated yet, but they are generated from junk DNA. A review on piRNA can be found here.

In a recent report scientists at Yale University have shown that piRNAs play pivotal role in regulating gene function. They discovered more than 13,000 Piwi-associated piRNAs in fruit flies. Out of these one was found to interact with Piwi, which finally binds to the chromatin and regulates the activity of the gene. Read the full story here.

via: Physorg

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rRNA

Ribosomal RNAs (rRNAs) constitute ribosomes where protein synthesis occurs. rRNA and ribosomal proteins combine to form ribosomes. Both prokaryotic and eukaryotic rRNA are made from longer precursors called preribosomal RNAs, or pre-rRNA .

In bacteria, a single 30S, 6500 nucleotides long, RNA precursor, after processing, makes 16S, 23S and 5S rRNAs. RNA at both ends of 30S precursor and between the rRNAs is removed during processing. The E. coli genome encodes 7 pre-rRNA molecules. All these genes have identical rRNA coding regions; but the regions in between the coding regions differ. The regions between the genes for 16S and 23S rRNA code for one or two tRNAs.

In eukaryotes, a 45S pre-rRNA transcript is processed to give rise to 18S, 28S and 5.8S rRNAs. The processing takes place in the nucleolus. The 5S rRNA is synthesized as a separate transcript.

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Poly(A) tail

At the 3′ end, most eukaryotic mRNAs have a string of 80-250 adenylate residues called the poly(A) tail. The poly(A) tail is added in a multistep process after the mRNA transcript has been synthesized. Two sequences are required for cleavage and polyadenylation (addition of ploy(A) tail) of the mRNA: 1) A highly conserved polyadenylation signal sequence AAUAAA, found 10 to 30 nucleotides upstream (on the 5′ side) of the cleavage; 2) A less well-defined sequence rich in G and U or U residues only, found 20 to 40 nucleotides downstream of the cleavage site. The cleavage generates the free 3′-OH group that defines the end of the mRNA, to which adenylate residues are added. Following proteins are required for cleavage and polyadenylation of pre-mRNAs.

1) Cleavage and polyadenylation specificity factor (CPSF): It is 360 kDa large complex made up of 4 polypeptides. It forms an unstable complex with the AAUAAA sequence.

2) Cleavage stimulatory factor (CStF1): 200 kDa heterotrimer.

3) Cleavage Factor I and II (CFI and CFII)

After CPSF has bound to the AAUAAA on the pre-mRNA, the CStF1, CFI, and CFII bind to the CPSF-mRNA complex. Interaction between CStF and GU or U-rich less well-defined sequence stabilizes the multiprotein complex. Finally, polyadenylate polymerase (poly(A) polymerase or PAP) binds to the complex before the cleavage can occur. The PAP binding links cleavage and polyadenylation, so that the free 3′ end generated after cleavage is rapidly polyadenylated. The assembly of this large multiprotein cleavage-polyadenylation complex around the AAUAAA signal in a pre-mRNA is analogous in many ways to the formation of the transcription-initiation complex at the TATA box.

Following cleavage at the poly(A) site, polyaenylation proceeds in two phases. Addition of the first 12 or so A residues occurs slowly, followed by a rapid addition of upto 200-250 A residues. The rapid phase requires the binding of multiple copies of poly(A) binding protein (PABII). PABII stimulates polymerization of additional A residues by PAP. PABII is also responsible for signaling PAP to terminate polymerization when te poly(A) tail reaches a length of 200-250 residues.

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The 5′ end of eukaryotic mRNA is capped with a guanine nucleotide (which is methylated forming 7-methyguanosine). The cap (5′-G) is added to the mRNA after transcription. The addition of 5′ G is catalyzed by a nuclear enzyme, guanylyl transferase. The cap is linked to the 5′ terminus of the mRNA through an unusual 5′,5′-triphospahe linkage. The 5′ cap is formed by condensation of a molecule of GTP with the triphosphate at the 5′ end of the transcript. The guanine is subsequently methylated at N-7 to form 7-methylguanosine. Additional methyl groups are added to the 2′ hydroxyls (-OH) of the first and second nucleotides adjacent to the cap. The methyl groups are derived from S-adenosylmethionine.

At the 3′ end, most eukaryotic mRNAs have a string of 80-250 adenylate residues called the poly(A) tail.

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All mRNAs contains two regions. A coding region which consists of a series of triplet codons representing the amino acid sequence of the coded protein, starting with and AUG (initiation codon) and ending with a termination codon. This is referred to as the open reading frame (ORF). Extra regions are present at both the 5′ and 3′ ends of an mRNA. The sequence at the 5′ end, preceding the initiation codon, is the leader sequence. The sequence following the termination codon, at the 3′ end, is called a trailer. They are also referred to as 5′ and 3′ untranslated regions, respectively. The UTR sequences are part of the transcription unit, but these are not used to code for the protein.

Eukaryotic mRNA constitutes only a small proportion of the total cellular RNA (approx. 3%). The half-life of mRNA in the yeast is short, ranging from 1-60 minutes. The mRNA is highly stable in higher eukaryotes: animal cell mRNAs have half-lives ranging from 4-24 hours.

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mRNA

mRNA encodes the amino acid sequence of the polypeptide specified by a gene. The function of mRNA is the same in all cells, but there are some differences in the synthesis and structure of prokaryotic and eukaryotic mRNA.

1) In bacteria, mRNA is transcribed and translated in the single cellular compartment, i.e, there is no spatial separation of the two processes. These two process in bacteria are closely linked and occur simultaneously. Translation starts, and ribosomes attach to 5′ end of bacterial mRNA, even before the transcription is complete. Bacterial mRNA is usually unstable, and translated into proteins for only a few minutes.

2) In a eukaryotic cell, however, mRNA transcription and translation occur in nucleus and cytoplasm, respectively. Thus the two process are spatially separated. The synthesis and maturation of mRNA occur exclusively in the nucleus. After these events, the mRNA is exported to the cytoplasm, where it is translated. Eukaryotic mRNA is highly stable and continues to be translated for several hours. However, many unstable mRNAs are known in eukaryotes.

The rate of transcription and translation in bacteria are similar. At 37°C, mRNA transcription occurs at a rate of approximately 40 nucleotides/second, which is very close to the rate of protein synthesis, roughly 15 amino acids/second. Thus it takes around 2 minutes to transcribe and translate an mRNA of 5000 bp, corresponding to an 80 kDa protein. 

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