In animals, microRNAs (miRNAs) bind to the 3′ UTRs of their target mRNAs and interfere with translation, although the exact mechanism of inhibition of protein synthesis remains unclear. Functional miRNA-binding sites in the coding regions or 5′ UTRs of endogenous mRNAs have not been identified. We studied the effect of introducing miRNA target sites into the 5′ UTR of luciferase reporter mRNAs containing internal ribosome entry sites (IRESs), so that potential steric hindrance by a microribonucleoprotein complex would not interfere with the initiation of translation. In human HeLa cells, which express endogenous let-7a miRNA, the translational efficiency of these IRES-containing reporters with 5′ let-7 complementary sites from the Caenorhabditis elegans lin-41 3′ UTR was repressed. Similarly, the IRES-containing reporters were translationally repressed when human Ago2 was tethered to either the 5′ or 3′ UTR. Interestingly, the method of DNA transfection affected our ability to observe miRNA-mediated repression. Our results suggest that association with any position on a target mRNA is mechanistically sufficient for a microribonucleoprotein to exert repression of translation at some step downstream of initiation.

Herpes Virus MiRNA Modulation: Viruses use a number of strategies to manipulate the cells of their host to ensure a successful infection. Herpesvirus saimiri (HVS) generates highly conserved small noncoding RNAs HSUR 1 and HSUR 2 , which modulate expression of a number of proteins in infected primate T cells. Cazalla et al. (p. 1563 ; see the Perspective by Pasquinelli ) observed complementarity between HSUR sequences and the seed regions of three different miRNAs—miR-142-3p, miR-27, and miR-16—and found that these HSURs could bind to the miRNAs. Furthermore, the level of mature miR-27 was modulated by binding to HSUR 1 , which targeted the miRNA for degradation.

Proteins are transported into and out of the cell nucleus via specific signals. The two best-studied nuclear transport processes are mediated either by classical nuclear localization signals or nuclear export signals. There also are shuttling sequences that direct the bidirectional transport of RNA-binding proteins. Two examples are the M9 sequence in heterogeneous nuclear ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of which appear to contribute importantly to the export of mRNA to the cytoplasm. HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3′ untranslated regions and has been shown to shuttle between the nucleus and cytoplasm (18, 19). We have identified in HuR a shuttling sequence that also possess transcription-dependent nuclear localization signal activity. We propose that HuR first may bind AU-rich element-containing mRNAs in the nucleus and then escort them through the nuclear pore, providing protection during and after export to the cytoplasmic compartment.

We have identified gas5 (growth arrest-specific transcript 5) as a non-protein-coding multiple small nucleolar RNA (snoRNA) host gene similar to UHG (U22 host gene). Encoded within the 11 introns of the mouse gas5 gene are nine (10 in human) box C/D snoRNAs predicted to function in the 2′-O-methylation of rRNA. The only regions of conservation between mouse and humangas5 genes are their snoRNAs and 5′-end sequences. Mapping the 5′ end of the mouse gas5 transcript demonstrates that it possesses an oligopyrimidine tract characteristic of the 5′-terminal oligopyrimidine (5′TOP) class of genes. Arrest of cell growth or inhibition of translation by cycloheximide, pactamycin, or rapamycin—which specifically inhibits the translation of 5′TOP mRNAs—results in accumulation of the gas5 spliced RNA. Classification of gas5 as a 5′TOP gene provides an explanation for why it is a growth arrest specific transcript: while the spliced gas5 RNA is normally associated with ribosomes and rapidly degraded, during arrested cell growth it accumulates in mRNP particles, as has been reported for other 5′TOP messages. Strikingly, inspection of the 5′-end sequences of currently known snoRNA host gene transcripts reveals that they all exhibit features of the 5′TOP gene family.