Polyadenylic Acid Sequences: Role in Conversion of Nuclear RNA into Messenger RNA

Darnell, James; Philipson, Lennart; Wall, Randolph; Adesnik, Milton

doi:10.1126/science.174.4008.507

Cited by 566 publications

(205 citation statements)

References 23 publications

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“…Because cordycepin interferes with polyadenylation, it was concluded that the probe must have been hybridized in the untreated cells. However, cordycepin has not been used in previous studies on mammalian cell nuclear RNA metabolism for more than 1-3 h (Siev et al, 1969;Penman et al, 1970;Darnell et al, 1971;Mendecki et al, 1972) because of severe toxicity effects. Moreover, it has been clearly established that cordycepin inhibits the production of cytoplasmic mRNA in HeLa cells within 25-40 min (Penman et al, 1970).…”

Section: Discussionmentioning

confidence: 99%

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

Politz

Tuft

Prasanth

et al. 2006

MBoC

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Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy. INTRODUCTIONNuclear speckles are morphologically distinct regions of the nucleoplasm that contain pre-mRNA splicing components as well as poly(A) RNA (Carter et al., 1993;Zhang et al., 1994;Lamond and Spector, 2003). They are operationally defined by their immunostaining with a variety of pre-mRNA splicing factor antibodies, and they also show in situ hybridization signal using probes for poly(A). When these sites are viewed in the electron microscope, most of them are found to represent interchromatin granule clusters (Fakan et al., 1984. However, RNA polymerase II transcription sites are distributed throughout the nucleus, indicating that although some of the RNA present in interchromatin granules/speckles is nascent, transcription is not restricted to this compartment, and much of the poly(A) RNA there has likely been transcribed previously and/or at distant sites (Fakan and Nobis, 1978;Wansink et al., 1993, Zeng et al., 1997, Neugebauer and Roth, 1997, Cmarko et al., 1999Guillot et al., 2004). Indeed, although transcripts that are being rapidly produced or that contain many introns are sometimes observed in speckles at the light microscopy level (Johnson et al., 2000;Shopland et al., 2003), the majority of studies over the years has indicated that most speckles are not primary sites of pre-mRNA splicing (for reviews, see Mattaj, 1994;Huang and Spector, 1996a;Neugebauer and Roth, 1997;Lamond and Spector, 2003). In two cases, a single species of pre-mRNA has been followed from its transcription site to the nuclear pore, and in neither case did the RNA seem to accumulate in nuclear subcompartments (Singh et al.,...

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Section: Discussionmentioning

confidence: 99%

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

Politz

Tuft

Prasanth

et al. 2006

MBoC

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“…One of our early experiments that suggested a lag before the addition of some of the poly(A) segments involved the use of actinomycin (2,4,31). This drug stops the synthesis of hnRNA, but for a brief period allows presumably complete ImRNA molecules to acquire poly(A) within the nucleus.…”

Section: Effects Of Actinomycin On Poly(a) Synthesismentioning

confidence: 99%

Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis.

Salditt-Georgieff¹,

Harpold²,

Sawicki³

et al. 1980

The Journal of Cell Biology

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A kinetic analysis of the appearance of [3H]uridine label in RNA sequences that neighbor poly(A), as well as the incorporation of [3H]adenosine label into both the RNA chain and the poly(A) of poly(A)-containing molecules, shows that poly(A) is added within a minute or so after RNA chain synthesis in Chinese hamster ovary cells and HeLa cells. Previous conclusions by several groups (5-7) that poly(A) might be added as long as 20-30 min after RNA synthesis appear to be in error, and the present conclusion seems much more in line with several different types of recent studies with specific mRNAs that suggest prompt poly(A) addition (13-16).

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“…2 shows the superimposed electrophoretic patterns of polyribosome-associated [3H]adenosine labeled RNA from free and membrane-bound ribosomes and of the respective RNAase digests. A peak with the electrophoretic mobility described for poly-A [1] was observed in both the RNAase digests of free and membrane-bound polyribosomal RNA. By contrast, no RNAase resistant material appeared in the digests of [3H]uridine labeled RNA analyzed in the same way.…”

Section: Resultsmentioning

confidence: 73%

“…see the review by Darnell et al [1]). Poly-A sequences have also been found in mRNA of both DNA viruses [2,3] and RNA viruses [4][5][6].…”

Section: Introductionmentioning

confidence: 96%

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Presence of polyadenylic acid sequences in RNA of membrane‐bound polyribosomes

1972

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Polyadenylic Acid Sequences: Role in Conversion of Nuclear RNA into Messenger RNA

Cited by 566 publications

References 23 publications

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis.

Presence of polyadenylic acid sequences in RNA of membrane‐bound polyribosomes

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