miRNA

Published online before print March 7, 2006, 10.1073/pnas.0601268103PNAS | March 14, 2006 | vol. 103 | no. 11 | 3951-3952Institution: Bolivia: PNAS Sponsored

Commentary:MicroRNAs: New players in an old gameMalavika Gupta, and Gary Brewer*Department of Molecular Genetics, Microbiology, and Immunology,University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, PiscatawayNJ 08854

One of the cardinal steps in regulating gene expression is mRNA decay, and the numerous pathwaysand mechanisms that exist to regulate it underscore its importance.mRNA decay is regulated by trans-acting factors that assemble on cis-acting elements (1, 2).Together, they serve to up- or down-regulate a given mRNA. Some of the mechanisms thatregulate mRNA levels involve surveillance pathways such as nonsense-mediated decay (NMD)and nonstop decay (NSD).The NMD (nonsense-mediated decay ) pathway limits accumulation of mRNAs that contain apremature termination codon and whose translation would produce a truncated protein.In NSD (nonstop decay ), mRNAs that do not contain a termination codon because of improperpoly(A) site selection within the coding region are rapidly degraded by the exosome,a complex of 3'->5' exoribonucleases (3). Other pathways involve recognition of 3' UTRsequences by specific RNA-binding proteins.For example, in AU-rich element (ARE)-mediated mRNA decay (AMD), binding of specific ARErecognition proteins to the 3' UTR initiates mRNA degradation (1, 4).

To one degree or another, all these decay pathways involve the step-wisedeconstruction of a mRNA involving 3'->5' trimming of the poly(A) tail,a process referred to as deadenylation; this is followed by removal of the5' m7GpppG cap and both 5'->3' and 3'->5' degradation of the mRNA body (5–7).This step-wise mechanism, first elucidated in Saccharomyces cerevisiae,has been recognized for some time now.Another mRNA decay pathway that has garnered much attention lately isRNA interference (RNAi).

First discovered in Caenorhabditis elegans (8), RNAi has now been observedin several other multicellular organisms, including mammals. RNAi is triggeredeither by a small interfering RNA (siRNA) or, in some cases, by a microRNA (miRNA)that induces mRNA degradation via endoribonucleolytic cleavage within the site ofsi/miRNA–mRNA annealing. siRNAs derive from sources such as double-stranded RNA,transposons, and viruses and are perfectly complementary to their mRNA targets (9–11).

miRNAs are {approx}22 nt in length and are encoded within the genomes of bothplants and animals. miRNAs contain regions possessing imperfect complementarityto 3' UTRs of mRNA subsets to which they anneal. This leads to translationalsilencing to posttranscriptionally control gene expression (12). In this issue of PNAS, Wu et al. (13) demonstrate that a miRNA can also promoterapid mRNA degradation by accelerating the initial rate-limiting step, deadenylation.

The life of a miRNA begins as a miRNA precursor called primary miRNA (pri-miRNA). In metazoans, the enzyme Drosha catalyzes the first cleavage event that results inthe production of a pre-miRNA intermediate. With the assistance of Exportin5,this intermediate travels to the cytoplasm, where it undergoes further cleavageby a second enzyme called Dicer. Cleavage by Dicer leaves an RNA duplex that isunwound, and the so-called guide strand, which contains complementarity tomRNA targets, assembles with Argonaute proteins and others to form the RNA-inducedsilencing complex (RISC) (14).

A large body of evidence indicates miRNAs to be translational repressors and siRNAsto be purveyors of mRNA degradation. A few exceptions to translational silencingby miRNAs have begun trickling into the literature, however. For example,mammalian miR196a is perfectly complementary to one of its target transcripts,HOXB8, except for a single G:U wobble base pair (Fig. 1) (11).

miR196a directs endoribonucleolytic cleavage of HOXB8 mRNA, which encodesone member of a group of related transcription factors involved in animal development.

Although miR196a exhibits near-perfect complementarity to its target mRNA,miR125b and the miRNA known as let-7, do not (Fig. 1) (15).Although Wu and Belasco (15) demonstrated that miR125b target associationdid repress translation, this miRNA surprisingly led to reduced mRNA levels as well. In work presented here, they demonstrate that miR125b and let-7 increase mRNAdecay rates upon association with their target mRNAs, not by endoribonucleolyticcleavage but rather by promoting rapid deadenylation.

transcriptional pausingInstitution: Bolivia: PNAS SponsoredPublished online before print March 13, 2006, 10.1073/pnas.0600508103PNASMarch 21, 2006 | vol. 103 | no. 12 | 4439-444

biologicla science/biophysics:Thermodynamic and kinetic modeling of transcriptional pausingVasisht R. Tadigotla, Dáibhid Ó Maoiléidigh, Anirvan M. Sengupta, Vitaly Epshtein,Richard H. Ebright, Evgeny Nudler, and Andrei E. Ruckenstein

The basis for our quantitative analysis is the structural and mechanistic modelof the elongation complex (EC), sketched in Fig. 1.The EC consists of a melted DNA duplex region of 12–14 nt (transcription bubble)enclosed within RNAP and stabilized by interactions with the enzyme and with thelast 8 or 9 nt of the synthesized RNA transcript (the DNA–RNA hybrid). The RNAtranscript upstream of the hybrid exits RNAP via the "RNA exit channel";whereas the duplex DNA downstream of the bubble threads through a "sliding clamp”in the enzyme, which holds on tightly to the DNA while allowing for smooth sliding during transcription elongation.

The "secondary channel" provides the access of the incoming nucleoside triphosphate(NTP) to the active center where the catalysis of phosphodiester bond formation takesplace, resulting in the elongation of the RNA transcript by one nucleotide. Immediatelyafter the transcript elongation step the EC is in the so-called "pretranslocated" state(translocational state 0) in which the 3' end of the transcript overlaps the catalytic site.The next incorporation step requires that RNAP translocate forward by one base pair,into the "posttranslocated" state (translocational state +1),making the catalytic center available for the binding of the next NTP.

transposonsInstitution: Bolivia: PNAS SponsoredPublished online before print March 14, 2006,March 21, 2006 | vol. 103 | no. 12 | 4540-4545

biological science/geneticsSelf-synthesizing DNA transposons in eukaryotesVladimir V. Kapitonov*, and Jerzy Jurka*

Genomes of most eukaryotes are populated by DNA copies of parasitic elementsknown as transposable elements (TEs) capable of reproducing themselves inthe host genome in a non-Mendelian fashion (1, 2). Understanding the biologyof transposable elements is of great importance because of their increasinglywell documented impact on the host genome (2, 3). Moreover, transposable elementscan be used as powerful tools in genetic engineering (4). Despite an enormousdiversity of eukaryotic TEs, they belong to only two types, called retrotransposonsand DNA transposons. Whereas a retrotransposon is transposed (retroposed) viareverse transcription of its mRNAs, a DNA transposon is transposed via transferof its genomic copy from one site to another.

Each type includes different classes and families of TEs composed of autonomousand nonautonomous elements. Whereas an autonomous element encodes a completeset of enzymes characteristic of its family, a nonautonomous element encodes none,or only some of them, and depends on enzymes encoded by its autonomous relative. Transposition of a retrotransposon is catalyzed by reverse transcriptase andendonuclease (EN) domains of a polyprotein encoded by itself or by otherretrotransposons. All retrotransposons can be further divided into two subclassescalled LTR and non-LTR retrotransposons (5).In addition to the reverse transcriptase/EN polyprotein, most non-LTRretrotransposons code for a second protein characterized by poorly understoodactivities, including RNA/DNA binding, chaperone, and esterase.An mRNA molecule expressed during transcription of the genomic non-LTRretrotransposon is reverse transcribed and inserted in the genome (5).LTR retrotransposons, including endogenous retroviruses, represent the mostcomplex TEs in eukaryotes. An LTR retrotransposon may carry three ORFs codingfor the gag, env, and pol proteins, the latter is composed of the reverse transcriptase,EN, and aspartyl protease domains (5).The endonuclease domain in LTR retrotransposons is usually called integrase (INT)and is distantly related to the DDE transposases (TPase) encoded by MarinerDNA transposon