Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human.

Post-transcriptional synthesis of 2¢-O-methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modi®cation in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and speci®cally targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D-H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body-speci®c localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5¢ and 3¢ hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism. Keywords: box C/D RNA/box H/ACA RNA/Cajal body/ RNA localization/scaRNA IntroductionIn eukaryotes, most aspects of the nuclear biogenesis of cellular RNAs can be linked to distinct subnuclear domains, also called nuclear organelles (Lamond and Earnshaw, 1998;Misteli and Spector, 1998;Lewis and Tollervey, 2000). The structural and functional compartmentalization of the nucleus requires speci®c targeting of many RNAs to their correct destination. Therefore, intranuclear RNA transport plays a fundamental role in eukaryotic gene expression. However, in contrast to the extensively studied nucleo-cytoplasmic RNA transport, little is known about the principles governing RNA localization within the nucleus. In this study, we investigate the mechanism directing speci®c localization of small Cajal body (CB)-speci®c RNAs (scaRNAs) to the nucleoplasmic CBs.CBs are evolutionarily conserved nucleoplasmic organelles that contain many protein and ribonucleoprotein (RNP) factors involved in mRNA and rRNA biogenesis (Bohmann et al., 1995;Matera, 1999;Gall, 2000;Dundr and Misteli, 2001;Ogg and Lamond, 2002). The list of proteins and RNPs detected in CBs includes RNA polymerase I, II and III subunits, basal transcription factors, small nuclear RNPs (snRNPs) mediating mRNA splicing and small nucleolar RNPs (snoRNPs) required for rRNA modi®cation and nucleolytic processing. CBs, however, do not contain nascent mRNAs and rRNAs, indicating that neither synthesis nor processing of these RNAs occur in this organelle. Instead, the available data are most consistent with the idea that the major function of CBs is in maturation, assembly and/or traf®cking of snRNPs, snoRNPs and transcription complexes which later function in mRNA and rRNA biogenesis in other nuclear compartments (Gall et al., 1999;Sleeman and Lamond, 1999;Gall, 2001;Carmo-Fonseca, 2002;Ogg and Lamond, 2002;Verheggen et al., ...

Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.

Telomerase is a ribonucleoprotein enzyme that counteracts replicative telomere erosion by adding telomeric sequence repeats onto chromosome ends. Despite its well-established role in telomere synthesis, telomerase has not yet been detected at telomeres. The RNA component of human telomerase (hTR) resides in the nucleoplasmic Cajal bodies (CBs) of interphase cancer cells. Here, in situ hybridization demonstrates that in human HeLa and Hep2 S phase cells, besides accumulating in CBs, hTR specifically concentrates at a few telomeres that also accumulate the TRF1 and TRF2 telomere marker proteins. Surprisingly, telomeres accumulating hTR exhibit a great accessibility for in situ oligonucleotide hybridization without chromatin denaturation, suggesting that they represent a structurally distinct, minor subset of HeLa telomeres. Moreover, we demonstrate that more than 25% of telomeres accumulating hTR colocalize with CBs. Time-lapse fluorescence microscopy demonstrates that CBs moving in the nucleoplasm of S phase cells transiently associate for 10 -40 min with telomeres. Our data raise the intriguing possibility that CBs may deliver hTR to telomeres and/or may function in other aspects of telomere maintenance. INTRODUCTIONTelomeres are specific nucleoprotein complexes protecting the termini of eukaryotic linear chromosomes from degradation, end-to-end fusion and undesired recombination (for recent reviews, see Wong and Collins, 2003;Ferreira et al., 2004;Smogorzewska and de Lange, 2004;Blasco, 2005). In humans, the DNA component of telomere is made up of ϳ5-15 kb of double-stranded 5Ј-TTAGGG-3Ј repeats that terminates in a 3Ј overhang of ϳ50 -300 bases (Makarov et al., 1997;Wright et al., 1997). By invading into proximal double-stranded telomeric sequences, the single-stranded overhang supports formation of a large duplex loop, termed the T-loop (Griffith et al., 1999). Because of the unidirectionality of conventional DNA polymerases, the ends of telomeres cannot be fully duplicated and human telomeres lose ϳ50 -200 base pairs during each cell division cycle and eventually, critically short telomeres induce cell cycle arrest called proliferative senescence. Therefore, by imposing a limit on the replicative life span of somatic cells, telomere erosion represents an innate mechanism for tumor suppression (Smogorzewska and de Lange, 2004).The replicative telomere erosion can be balanced by the telomerase reverse transcriptase that adds telomeric DNA repeats to the 3Ј overhang of telomeres (reviewed in Collins and Mitchell, 2002;Cong et al., 2002). In humans, telomerase activity is not detectable in most somatic cells, but germ line and other highly proliferative cells as well as the majority of tumor-derived cell lines possess highly active telomerase, indicating that maintenance of telomere length is necessary for indefinite proliferation of human cells (Kim et al., 1994;Greider, 1996;Shay and Bacchetti, 1997). Telomerase is a ribonucleoprotein (RNP) enzyme that is minimally composed of the telomerase RNA (TR) that spec...