correlation of hnrnp structure and nascent transcript cleavage

Cell, Vol. 26, 155-l 65. October 1981 (Part 2). Copyright 0 1981 by MIT

Correlation of hnRNP Structure and Nascent Transcript Cleavage

Ann L. Beyer, Amy H. Bouton and Oscar L. Miller, Jr. Department of Biology University of Virginia Charlottesville, Virginia 22901

Summary

Using electron microscopy of spread chromatin, we have observed nonnucleolar transcription units from Drosophila melanogaster and Calliphora erythrocephala that display specific cleavage of nascent transcripts. We have quantitatively analyzed 20 of these relatively long transcription units. The primary RNP structure of homologous transcripts is nonrandom with respect to both RNA sequence and the cleavage event. In general, released RNA fragments have a smooth fibrillar RNP morphology (-50 A wide) and retained segments have a thicker particulate morphology (-250 A diameter). A characteristic secondary structure formation also accom- panies cleavage-that is, RNP fibril loops form by association of noncontiguous transcript sequences that correspond to the terminal regions of the segment to be released. RNP particles form at the loop base sequences prior to their association and apparently coalesce upon loop formation. These loops, and thus the released segments, range in length between 1 and 25 kb on the examples we have analyzed. Cleavage of nascent hnRNA transcripts appears to be a fairly common event in these organisms and occurs within 0.3-3 min after transcription of the cleavage site.

Chooi, 1976; Scheer et al., 19791, the process is not as well understood. We report here on analysis of hnRNA transcription units from two dipteran species, Drosophila melanogaster and Calliphora erythrocephala (blow fly), that exhibit specific cleavage of nascent transcripts. In agreement with Laird and Chooi (19761, we find that nascent transcript cleavage.is not uncommon on Drosophila embryo hnRNA transcription units and that some transcripts undergo more than one cleavage. Based on the size of these transcription units and the size of the released RNA fragments, it is possible that the cleavages correspond to cleavage at 3’ pre-mRNA termini or the release of functional domains from polycistronic transcription units, or both.

Previously we have shown by electron microscopy of spread Drosophila chromatin that the primary RNP structural features of nascent hnRNA transcripts are nonrandom and sequence-dependent. Specifically, the nascent transcripts occurred in either a smooth RNP fibrillar form or an RNP particle form, and the choice between the two structural types was determined by the underlying RNA sequence (Beyer et al., 1980). In analyzing transcription units that displayed nascent transcript cleavage, we particularly were in- terested in mapping hnRNP structure. We find a strong correlation between both primary and secondary hnRNP structural features and the cleavage process. This is direct evidence that the proteins associated with nascent hnRNA are distributed in a functionally meaningful way. The hnRNP configurations accom- panying cleavage are identical in the two organisms and at different developmental stages, and are inform- ative regarding the mechanics of the cleavage process.

Introduction Results

Specific endonucleolytic cleavages are a common feature of posttranscriptional RNA processing path- ways (reviewed by Abelson, 1979). Some of these cleavages occur on nascent RNA molecules, as can be seen when chromatin is dispersed for electron microscopic visualization (Miller and Hamkalo, 1972; Laird and Chooi, 1976; Scheer et al., 1979; Grainger and Maizels, 1980). In chromatin spreads, transcription units are identified as arrays of lateral RNP fibrils extending from a DNP axis (Miller and Beatty, 1969). These transcription units, both nucleolar and nonnucleolar, typically exhibit regularly increasing gradients of RNA fibril length, consistent with the concept of a specific transcription initiation site and completion of transcription before RNA cleavage. Some transcription units, however, display abrupt changes in RNP fibril length, suggesting that cleavage of RNA occurs at specific sites on nascent transcripts. For ribosomal genes, such cleavages correspond to the enzymatic separation of large and small subunit rRNA precursors (Miller and Hamkalo, 1972; Grainger and Maizels, 1980). For nonribosomal transcription units (Laird and

The transcription units analyzed are nonnucleolar transcription units from embryos and fat body tissues of D. melanogaster and C. erythrocephala. Such transcription units vary considerably in length and in RNP fibril density, as expected for hnRNA coding regions. Criteria for distinguishing nucleolar and nonnucleolar transcription units in this type of electron microscopic preparation have been described previously (Foe et al., 1976; McKnight and Miller, 1979).

The transcription units analyzed in this report display complex fibril length distributions that we interpret as resulting from cleavage of nascent transcripts. That is, the RNP fibril length gradients display abrupt transitions resulting in two or three gradients of fibril length in a group of closely spaced transcripts. The short fibrils remaining after the cleavage generally are longer than the very short fibrils characteristic of initiation sites. Examples of these discontinuous fibril length distributions are shown as insets in Figures 1 c-4c.

Although the possibility that the multiple fibril length

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gradients are the result of independent promoters rather than of nascent transcript cleavage events cannot rigorously be excluded, we agree with Laird and Chooi (1976) in their arguments for the implausibility of the individual promoter hypothesis. First, this alternative predicts short transcripts between the putative second promoter and the putative first termination site, and such transcripts typically are not observed. Second, the independent promoter alternative re- quires gene-specific termination control for the over- lapping transcription units.

We have visualized about 70 transcription units that exhibit apparent nascent transcript cleavage. Twenty of these have been mapped and quantitatively analyzed (see Experimental Procedures). These 20 display a total of 28 cleavage events-that is, the fibrils of 12 of the 20 transcription units were cleaved at one site and the fibrils of eight were cleaved successively at two sites. The lengths of these transcription units range between 1.2-8.5 pm, or about 4.9-40.6 kb, with a mean of 21 .l kb (see Experimental Procedures for pm to kb conversion). Five of these transcription units are shown in Figures 1-4, and the RNP fibril maps of six others are shown in Figure 5a-5f.

The RNP fibril maps (Figures 1 c-4c and 5) display RNP structural features on RNA sequence-aligned transcripts (Figure 1; Beyer et al., 1980). The template-distal, or 5, ends of the nascent transcripts are to the left in the fibril maps, while the template-attached 3’ termini are to the right. In our previous report, we aligned RNA sequences by aligning 5’ termini at the left in these maps. However, because of the apparent 5’-end cleavages on the transcription units analyzed herein, we have aligned like sequences by abutting the 3’ termini of individual fibers to the appropriate position on a schematic “DNA axis” drawn as a dashed sloped line corresponding to the best-fit least squares line to the first (uncleaved) fibril gradient. Transcript sequences are not aligned per- fectly by these procedures because of variation in fibril compaction (that is, RNA content per unit length) from one fibril to the next. The chromatin spreading procedure disrupts the compact tertiary structure of transcription complexes for display in a dispersed two-dimensional array; the variation in fibril compaction is inherent to the method, representing a variation in the degree of dispersal of each transcript. Thus RNP structural features, such as RNP particles, are not aligned exactly in the RNP fibril maps, but visual inspection reveals that they are located nonrandomly. A previous statistical analysis also indicated that the apparent variation in RNP particle location was within the inherent error of the method (Beyer et al., 1980).

The transcription unit in Figure 1 was obtained from a Drosophila embryo and exhibits ten fibrils. The last two fibrils apparently are cleaved. In the RNP fibril map, RNP particles, hairpin loops and fibril foldbacks that occur on the transcripts are indicated schemati-

tally at the appropriate positions. In agreement with our previous findings, RNP particles and hairpin loops occur nonrandomly, with respect to RNA sequence, on the transcripts of a particular transcription unit. Note that the RNA cleavage occurs at or near the same sequence on the two cleaved transcripts (#9 and #lOI and near the transition between smooth fibrillar and particulate regions. It is important to point out in regard to this and the following figures that previously we have demonstrated that RNA is not compacted preferentially in RNP particle form over RNP fibrillar form when prepared under these conditions (Beyer et al., 1980). We thus consider it very unlikely that the short thicker particulate fibrils represent a compacted or packaged full-length transcript. The estimated transcription unit length is 4.9 pm, or about 23 kb, and the length of the released RNP fragment is about 18 kb.

Note also in Figure 1 that the long fibrillar region that eventually is released apparently is involved in long-range intramolecular interactions as indicated by its entangled structure. In all but one of the 28 cleavage events we analyzed, the RNP that will be released appears folded back on itself in an RNP loop structure. In transcription units that do not display nascent transcript cleavage, the RNP fibrils typically do not exhibit secondary structure of this type. Similar loops have also been observed on RNP fibrils of amphibian oocyte lampbrush chromosome transcription units (Sommer- ville, 1981).

A transcription unit from a Calliphora embryo whose transcripts are cleaved successively at two specific sites is shown in Figure 2. RNP fibril loops that encompass the portion of RNA to be released occur before both cleavages. Note also that the loops occur at similar positions on different fibrils and thus that the loops and the particulate loop bases may contain specific RNA sequences. The first cleavage occurs in the middle of a mainly fibrillar region, very near a single particle at the downstream base of the first loop. The second cleavage is near the 3’ loop base site of the second loop, and near a fibrillar-particulate RNP transition. The estimated transcription unit length is 17.6 kb, the first released fragment is about 4 kb and the second released fragment is about 1.7 kb.

The transcripts of a third transcription unit, shown in Figure 3, are cleaved at a single site. This is an unusual example in that the fibrils are relatively defi- cient in both RNP particles and RNP loops. The cleavage occurs at the site of an RNP particle that appears on all of the more mature transcripts, but that forms a loop base on only one of them (#12). Two of the most mature transcripts are not cleaved (a phenomenon that is also seen in Figures 4, 5b and 5e), suggesting that cleavage is not required for continued transcription and that each transcript has an independent probability of being cleaved or acquiring competency for cleavage or both. The transcription unit is about

hnRNP Structure and Transcript Cleavage 157

b RNP length (pm) 0 1 2 3 \ \ \ \ \ \ \

\ \ \ 0 1 \ \ DNP tprn,

5

\ \ . . . . . .

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Figure 1, Analysis of Transcript Cleavage and Morphology

(a) Electron micrograph of a Drosophila nonribosomal transcription unit. Bar = 1 pm (b) Interpretive tracing of micrograph. (Dashed line) template chromatin strand: (solid lines) RNP fibrils; (0) RNP particles that occur on the fibrils. Transcripts numbered in order of increasing maturity: question marks indicate those in which the fibril path length was difficult to determine. The tracings shown of these fibrils are possible interpretations that are consistent with the micrograph. (c) RNP fibril map: graphic representation of linearized RNP fibrils. Spacing of the fibrils on the vertical axis corresponds to their position on the chromatin template. The dashed line represents the best-fit least squares line to fibril length for the first (uncleaved) fibril gradient (fibrils l-8 in this figure). (0) RNP particles; (cross-hatched regions) short lateral projections from the fibrils. interpreted as double-stranded hairpin loops. Dotted line drawn over the “looped-out” fibril segment connects points of intramolecular contact. Hairpin or open loops that occur within a larger loop are not indicated on the map because the 5’-3’ direction of the transcript in the loop is not known. (Inset) Graph of RNP fibril length as a function of DNP position.

18 kb long and the released RNA is about 13 kb long. The micrograph in Figure 4 shows two homologous

nonnucleolar transcription units on newly replicated sister chromatids. Such paired transcription units commonly are seen when the nuclei of early cellular blastoderm Drosophila embryos are disrupted in the late S or G2 phase of the synchronous cell cycle. Analysis has shown that they exhibit similar transcriptional activity (McKnight and Miller, 1979) and similar RNP structure (Beyer et al., 1980). As seen in the fibril maps (Figure 4c), the transcripts of these two units also undergo two similar nascent RNA cleavages. In transcription unit 4/3, RNP fibril loops that encompass the portion of RNA to be released form before both cleavages. As in Figure 2, the first cleavage occurs in the middle of a fibrillar segment near a single particle that occurs at the loop base. The second cleavage is near the downstream loop base and near a fibrillar-particulate transition. Both transcription units are about 27 kb in length; the first released

segments are about 4.5 kb in length and the second released segments are about 10.5 kb in length.

Some general features and ultrastructural corre- lates to the cleavage process can be noted from examination of these transcription units (Figures l-4) and the RNP fibril maps in Figure 5. First, transcript cleavage is almost always accompanied by RNP loop formation on the segment that will be released. The observed loops range in length between 0.1-3.8 pm, or about 0.9-24.8 kb, and average 9.6 kb. These RNP loops are of the smooth fibrillar RNP morphology except for the loop base (corresponding to the point of contact between noncontiguous transcript sequences), which generally appears as an RNP particle. These particulate loop bases occur at specific sites on the more mature transcripts immediately before cleavage. However, on less mature transcripts, the RNA may be folded back on itself at apparently random sequence sites. For example, fibrils 12’-17’ of Figure 4/3 exhibit such variable loop structures,

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C RNP length (urn) 0 1 2 \ ‘\ I 1

Figure 2. Analysis of Transcript Cleavage and Morphology on a Calliphora Nonribosomal Transcription Unit

Same as in Figure 1, Bar in (a) = 1 pm.

whereas fibrils 19’ and 21’ display presumably mature nonrandom loops. This “variable loop” phenomenon is common, occurring to some extent on 16 of the 20 analyzed transcription units (see Figures 1, 3, 5b, 5c, 5d and 5f), and may represent a progressive sliding of RNP fibrillar structures and/or sequences past each other until a “stable” loop structure is formed. RNP particles generally are not present at the base of variable loops. In some cases, the 3’ end of the stable loop base is not yet synthesized when variable loops are seen (as in Figure 4 example above), but in other cases both ends have been synthesized but are not in contact (see for example some of the transcripts in Figures 3 and 5f). RNP particles are often present at potential sites of stable loop formation even though no loops or only variable loops at other sites are present (see for example some of the transcripts in Figures 2, 3 and 5b, e and f). On some of the transcripts in Figures 4 and 5b, the eventual 5’ loop nucleation site is particulate even though the sequence that occurs at the 3’ loop base site is not yet synthesized. This is consistent with the notion that specific RNA sequence regions are responsible for the deposition of (specific?) RNP particle proteins, which then tend to as- sociate with another particulate site forming an RNP fibril loop. Once an RNP loop is formed, the loop base particles average 1 O-l 5% larger in diameter than other particles on the same transcription unit that do not occur at loop bases. The RNP particles that are not present at loop bases average about 250 8, in diameter, in agreement with our previous report (Be- yer et al., 1980).

The 5’ loop bases are very near, but often not at,

the 5’ ends of the transcripts. Thus when loops form, the short segment between the 5’ terminus and the 5’ loop base is excluded from the loop forming a ‘tail” (arrows in Figure 4d). These tails range in length between 0.01-0.35 pm (about 0.1-2.3 kb) and average 0.9 kb or 6% of total transcript length. The reso- lution of our method is insufficient to determine whether these tails are also removed at the time of cleavage or whether they are spliced onto the retained portion of the transcript. That is, it is not possible to distinguish between the loss or addition of 6% of the transcript length because of the variation in fibril compaction that is inherent to the method.

Unlike the portion that is released by cleavage that predominantly is fibrillar, the retained portion of the transcript can be either fibrillar or particulate. Based on the 20 transcription units analyzed for this report plus visual inspection of an additional 48, it was determined that 60% of the cleavage events result in a mainly particulate retained portion. When the retained portion is fibrillar, the cleavage occurs very near or at the single particulate site that corresponds to the loop base (see for example Figures 3 and 5f and the first cleavage in Figures 2 and 4). A second round of loop formation and cleavage is sometimes observed on the retained fibrillar portion (Figures 2 and 4). When the retained portion is particulate, the 5’-most particle of the closely spaced particle series forms the 3’ end of the loop base. Again, the subsequent cleavage is at or near the loop base. Occasionally, the cleavage site apparently is displaced slightly downstream into the middle of the particulate region (Figure 5b, rare), while in several others it apparently is displaced slightly


RNP length (pm) ’

. 2 l... ., -

.

Figure 3. Analysis of Transcript Cleavage and Morphology on a Drosophila Nonribosomal Transcription Unit

Same as in Figure 1. Bar in (a) = 1 pm.

upstream into the fibrillar region (Figures 1 and 5c, and second cleavage in Figure 4). This apparent dis- placement of the cleavage site from the loop base also confounds efforts to determine if tails are spliced or removed.

We have seen several cases of two successive cleavages occurring on nascent transcripts from both Drosophila and Calliphora. Up to three successive cleavages have been observed on Drosophila transcription units (Laird and Chooi, 1976). In our examples of two cleavages, the transcripts exhibit loop formation before both cleavages, but the first loop is removed before the second loop forms. In four of the eight cases we analyzed, it appears that the particulate sequence that is the 3’ loop base site for the first cleavage is also the 5’ loop base site for the second cleavage (Figures 2 and 4). In three others, there is a group of 2 to 3 closely spaced particles at or near the first cleavage site. It appears that the 5’-most particle of this group is the 3’ loop base for the first cleavage while the 3’-most particle is the 5’ loop base for the second cleavage (Figure 5e).

Thus in general, cleavage follows stable loop formation, which follows particle protein deposition at potential loop base sites. Loops form on the template distal ends of the transcripts on fibrillar regions whose length is demarcated by nearest-neighbor (but sepa-

rated) particles. Closely spaced particle series preferentially are excluded from loops, and loops apparently do not form between closely spaced particles.

There are exceptions to these general findings. RNP loops and particles generally do not appear at all sites on these transcripts that can be considered “potential” sites based on the structure of neighboring transcripts. We do not know if this reflects the efficiency of loop and/or particle formation or structural desta- bilization during chromatin spreading. (In this regard, Laird and Chooi [1976] analyzed a very similar cleavage phenomenon in Drosophila embryos using chromatin spreading for electron microscopy, but the fibrils in their micrographs do not display RNP loops.) In one of the 20 analyzed transcription units, cleavage occurs with no apparent loop formation, although near a fibrillar-particulate transition (Figure 5a). In the transcription unit in Figure 5d, a particle marks the cleavage site, but no “stable” loops form on the observed transcripts prior to cleavage. We have also seen several transcription units that exhibit loop formation and fibrillar-particulate transitions but no cleavage (not shown). In these cases, loops are generally seen only on one or two of the most mature transcripts that are nearing the transcription termination site. Perhaps cleavage occurs at these loop base sites after the transcripts are released.

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z RNP length (pm) 0 1 2 3 4

0 - i, 2 - \ \

l-45 \

. DNP (pm) \ \ 2. ....- - ,r’ - _ +, a . T \

3. \ \ \

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4. \ \ \ \ \ 4

z5 \ \ \

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1. ‘\ -45’ 0 2 4 6 \ , .

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Figure 4. Analysis of Transcript Cleavage and Morphology on Drosophila Nonribosomal “Sister-Chromatid” Transcription Units, Designated 4a and 4fl

(a), (b) and (c) as in Figure 1; arrow in (a) indicates a broken loop. (d) Higher magnification micrograph of three transcripts from transcription unit 4p. Fibrils 19’ and 21’ have an RNP fibril loop that has been removed from fibril 20’. Arrows indicate RNP “tails” (see text). Bars In (a) and (b) = 1 gm; bar in (d) = 0.5 pm.

Discussion assumed, the cleavages occur within 0.3-2.8 min (mean 1.7 min) after transcription of the cleavage site.

The Cleavage Process We have seen transcription units of this type in both We have analyzed nonnucleolar transcription units embryos and larval tissues (not shown) of the diptera, that display discontinuities in the RNP fibril length D. melanogaster and C. erythrocephela, and we have gradients, which we interpret to be the result of spe- no reason to suspect that they are peculiar to these cific endonucleolytic cleavage of nascent RNA tran- developmental stages or organisms. The same phe- scripts. Based on the length of the least mature nomenon previously has been noted in Drosophila cleaved transcript in each case, the cleavages occur embryo hnRNA transcription units (Laird and Chooi, when transcription has proceeded 0.8-8 kb past the 1976) and in amphibian oocyte lampbrush chromo- sequence at the cleavage site. If an approximate Pol some transcription units (Old et al., 1977; Scheer et II elongation rate of 3 kb/min (Kafatos, 1972) is al., 1979). It is difficult to estimate their frequency of


RNP length (urn)

I---

-y;

z

Figure 5. RNP Fibril Maps of Six Nonriboso- mal Transcription Units

All transcription units represented are from Drosophila embryos except (e), which is from a Calliphora embryo. RNP fibril mapping procedure described in Figure 1 and in text. To simplify this figure, positions of hairpin loops that occurred on the fibrils are not shown. RNP fibrils and DNP axes are drawn to the same scale except for the DNP axes in (b) and (e), which were expanded graphically to accomo- date the large number of RNP fibrils. The maps in (c). (e) and (f) represent portions that could be visualized clearly of presumably longer transcription units. The DNP axis labeling is based on extrapolated initiation sites.

+ cn 0 1 2 0 1 2 3 4

i - 011 d l,r :e

. .

60 t

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occurrence because many transcription units seen are transcribed too sparsely for meaningful analysis, but conservatively we would estimate that 25% of hnRNA transcription units display the abrupt transition in flbril length. Laird and Chooi (1976) obtained a similar frequency (4/l 2).

In the process that we visualize, the removal of an RNA segment from a nascent transcript typically is preceded by an intramolecular interaction between noncontiguous sequences, which correspond approx- imately to the termini of the released segment. The upstream interaction site is at or near the 5’ RNA terminus and the downstream site is at or near the site of RNA cleavage. We have examples of from 1 to 25 kb of RNA separating these sites. Presumably, these sites recognize each other and form a higher-order complex that is recognized by the appropriate en- zyme. Endonucleases involved in RNA metabolism, such as tRNA and rRNA maturation enzymes and splicing enzymes, typically recognize some secondary or tertiary structural feature rather than a primary RNA sequence (discussed by Abelson, 1979). The process that we visualize is similar at least superficially to the RNAase Ill-mediated removal of 16s and 23s rRNA precursors from the primary transcript of the E. coli

rRNA operon. Both ends of the precursor RNAs are generated by nuclease action on the double-stranded stems of stem-loop structures in which the 1.7 kb loop contains the 16s sequence and the 2.9 kb loop contains the 23s sequence (Young and Steitz, 1978; Bram et al., 1980). However, we do not know if RNA duplex formation occurs in the process that we visualize. The interaction at the base of the loop may be RNA-RNA (intramolecular or adaptor), protein-protein or some combination of the two. The occurrence of RNP particles at the interaction sites indeed suggests the involvement of protein in the recognition process. However, evidence indicates that RNA is exposed on the surface of hnRNP particles (reviewed by Martin et al., 1980; LeStourgeon et al., 1981) and that duplex regions in hnRNA are relatively protein- free (Calvet and Pederson, 1978). Regardless of the nature of the chemical interaction at the loop base site, that site presumably is specified by some aspect of RNA sequence. The identity and complexity of this sequence are a matter of speculation, especially since the cleavage process itself is understood poorly. If this putative sequence is contained within a single particle, it is presumably ho longer than about 500 nucleotides, which is the approximate length of RNA

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in an RNP particle under these preparative conditions (Beyer et al., 1980). We have examples where one RNP particle apparently functions as both an upstream and a downstream interaction site during two successive cleavages. This suggests that either there is no polarity to the interaction site or that 5’ and 3’ interaction sites occur near one another.

It is unlikely that the visualized process represents hnRNA splicing involved in intron removal. First, there is good evidence that splicing occurs on released, polyadenylated hnRNA molecules (reviewed by Lewin, 1980) and second, the multiple cleavages that we visualize are progressive in the order V-3’, which is not obligatory for intron removal (reviewed by Sharp, 1981). Perhaps the most serious objection, however, is the discrepancy in size between the released fragments and introns. Our data would predict that introns of dipteran species are very large and average ten times longer than exons (9 kb versus 0.9 kb-that is, mean loop length versus mean tail length), a situation that has not been seen in specific cloned Drosophila genes (reviewed by Spradling and Rubin, 1981). Some aspects of the cleavage process, however, are suggestive of the mechanism proposed for hnRNA splicing. The loop structures visualized are a predicted intermediate in splicing reactions that proceed via postulated snRNP adaptors (Yang et al., 1981). And, as in splicing, there is a progressive nature to the cleavage process. In cases of two successive cleavages, we have seen no transcripts that bypass the intermediate cleavage step. It has been suggested that the hnRNA splicing machinery binds to one end of the splicing site and translocates along the RNA until an acceptor end is recognized (Sharp, 1981). Our visualization of “variable loops” on transcript regions where “stable loops” will form indeed suggests such a sliding mechanism. A corollary to this aspect of the mechanism is that intermolecular association of loop bases is not expected, and again we have seen no cases of intermolecular interaction.

Correlation of hnRNP Structure and RNP Cleavage The ribonucleoprotein structure of the transcripts that are cleaved is nonrandom with respect to RNA sequence and with respect to the site of cleavage. In a previous electron microscopic study, we showed that nascent Drosophila hnRNA existed in one of two RNP forms (an RNP fibril about 50 A wide or RNP particles about 250 8, diameter) and that the distribution of these structures was determined by RNA sequence (Beyer et al., 1980). Here we show that in cases of nascent transcript cleavage, fibrillar RNP regions con- stitute the loops that are released, and particles or particulate regions demarcate loop termini but do not occur in loop structure. This correlation of hnRNP structure and a specific RNA cleavage is direct evidence that the proteins associated with hnRNA are

distributed in a functionally meaningful way and are involved in some type of RNA processing.

The RNP particles that we visualize on nascent transcripts are the same size as, and presumably correspond to, the 3OS-40s hnRNP particles that can be isolated from a wide variety of cell types (Georgiev and Samarina, 1971; Martin et al., 1974; Beyer et al., 1977). Antibodies to mammalian 30s hnRNP particle proteins bind to the template-attached RNP of amphibian oocyte lampbrush chromosomes (Martin et al., 1980). There are several lines of evidence indicating that, with the exception of double-stranded regions and poly(A) tails, hnRNA is nonspecifically packaged into repeating RNP particles of this size as it is transcribed (reviewed by Martin et al., 1980). The most recent corroborative evidence for the generality of RNP particle packaging is the finding that essen- tially all sequences of polyoma virus late transcripts, including introns and exons, occur in 30s particle form (Steitz and Kamen, 1981). Perhaps the most likely explanation for the discrepancy between these findings and our results is that the electron microscope preparative method, which disrupts native structure for display in a dispersed two-dimensional array, reveals differences in RNP stability. (Indeed, the hnRNP structure that we see is apparently only semi-stable under these conditions, as indicated by a degree of variability in hnRNP structure from one transcript to the next on a given gene.) The particulate and fibrillar structures may in fact be different forms of the same nucleoprotein complex. Reconstitution experiments with the major hnRNP particle protein of Artemia salina and isolated RNA molecules suggest that the same protein, at different protein:RNA ratios, is responsible for both the smooth RNP fibrillar structure and the repeating RNP particle structure (Nowak et al., 1980; Thomas et al., 1981). Regardless of whether the hnRNA of Drosophila and Calliphora is differentially packaged, or differentially stable after common packaging, we conclude that the resulting differential hnRNP structure is recognized in vivo and demarcates functional “domains” of the transcript. Biochemical evidence indicating functionally meaningful variation in hnRNP structure (that may or may not be related to our observations) is the somewhat greater stability to RNAase digestion of premessage sequences versus intervening sequences in hnRNP particles (Steitz and Kamen, 1981).

Loop formation appears to occur by the coales- cence of two separate RNP particles to form one particle. Do the RNP particles that eventually form loop bases differ in RNA and protein composition from those that do not? There are no obvious differences between the two classes in size, shape or time of deposition on nascent hnRNA. (The coalesced loop base particles, however, average 1 O-l 5% larger in diameter.) It is possible that a “loop base” RNA se-


quence specifies a characteristic loop base particle, which then seeks similar or complementary RNP particles for loop formation. Alternatively, there may be no specific sequences at the loop bases but rather a certain class of sequences that punctuate transcript “domains” and that stably bind RNP particle proteins. The rules for cleavage then would be to remove intact all (fibrillar) sequences that fall between punctuation sites. If the punctuation site is long or repetitive, resulting in a run of closely spaced particles, then only the outermost particle serve as loop base sites. The two models differ in the predicted degree of hetero- geneity of the particle population. There is also con- flicting evidence in the literature as to whether isolated hnRNP particles are homogeneous or heterogeneous in size, shape and protein composition (reviewed by Martin et al., 1980). The first model, wherein loop base particles are different from non-loop base particles, is more consistent with our observation that many transcription units with particulate transcripts show no evidence of loops or cleavage. However, loop base particles may differ only as a result of a modification of typical core RNP particles, for example, by the addition of an snRNP complex (Deimel et al., 1977; Lerner and Steitz, 1979; Zeive and Penman, 1981) or by protein methylation (Beyer et al., 1977) or phosphorylation (Martin et al., 1974; Gallinaro- Matringe et al., 1975).

Implications Regarding Transcription Unit Size and Structure Although only a limited number of transcription units exhibiting nascent transcript cleavage have been analyzed, it appears that they may be longer than nonnucleolar transcription units in general. The mean length of the 16 complex gradient transcription units that we analyzed from Drosophila cellular blastoderm stage embryos was 4.6 pm (or about 21 kb), whereas in a previous study from this lab, the mean length of 194 nonnucleolar transcription units from the same Drosophila stage was 2.9 pm (or about 11 kb) (Mc- Knight and Miller, 1976). Laird and Chooi (1976) also noted a length difference between complex gradient transcription units (R = 31 kb, N = 4) and simple gradient transcription units (ii = 18 kb, N = 8) of Drosophila embryos. All of these estimates of transcription unit size based on electron microscopic visualization are significantly larger than the same estimates based on isolated nuclear RNA size in Dro- sophila. That is, although a small proportion of Dro- sophila hnRNA sediments at 55S-60s on sucrose gradients (corresponding to transcripts of at least 20 kb), the average hnRNA molecule is 4-6 kb in length (Lengyel and Penman, 1975; Lamb and Laird, 1976). The mean length of the RNA segments that are released by nascent transcript cleavage is closer to reported hnRNA size: 9 kb (our results) and 7.8 kb

(Laird and Chooi, 1976). If these are pre-mRNA molecules, they are still significantly longer than the typical l-2 kb Drosophila mRNA (Lengyel and Penman, 1975; Zimmerman et al., 1980).

There is no simple explanation for the significant discrepancy in mean transcription unit length estimates when determined by electron microscopic visualization versus sucrose gradient sedimentation of early transcription products. Such a discrepancy has also been noted after electron microscopic studies of sea urchin transcription (Busby and Bakken, 1979) and amphibian oocyte transcription (Sommerville, 1981). In addition, the fairly common occurrence of nascent transcript cleavage, as described here, is not accomodated in generally proposed models of transcription unit organization. It is possible that the two observations are related, in that endonucleolytic processing of RNA before or very soon after transcriptional termination precludes the efficient isolation of primary transcription products (discussed by Laird and Chooi, 1976; Lamb and Laird, 1976). In specific examples where transcription unit initiation and termination sites have been investigated rigorously, it has been shown that some 3’ pre-mRNA termini are generated by cleavage rather than by transcriptional termination. Transcription past poly(A) sites occurs in several adenovirus and SV40 transcription units (reviewed by Ziff, 1980) in the mouse p globin gene (Hofer and Darnell, 1981) and perhaps also in the mouse immunoglobulin S gene (Maki et al., 1981). Cleavage at these sites apparently occurs on nascent transcripts of the Ad2 major late transcription unit (Nevins and Darnell, 1978) and perhaps also very early on /I globin transcripts, as suggested by the controversy concerning recovery of larger than 1% “primary” transcripts (Hofer and Darnell, 1981). Messenger regions of the human mitochondrial genome are also released posttranscriptionally from a large primary transcript by endonucleolytic action on the ends of tRNA gene sequences that punctuate the transcript, separating different mRNA and rRNA coding regions (Ojala et al., 1980). As far as we know, however, there is no evidence to indicate that either poly(A) site cleavage or tRNA site cleavage depends on the involvement of a noncontiguous upstream sequence. The loops, rather, are suggestive of inverted repeats in the transcripts. (Perhaps some of the abundant transcripts of cop&like insertion elements [Finnegan et al., 19781, which are flanked by short inverted repeats in addition to longer direct repeats [Levis et al., 19801, arise by posttranscriptional release from larger transcripts.)

In considering the large size of the visualized transcription units, it should be noted that their mean length (21 kb) is about the size of an average band or chromomere in Drosophila. It was suggested originally that this unit represented one gene (Bridges, 1935)

Cell 164

but analysis of mRNA complexity in Drosophila larvae indicates that there are three times more coding regions represented in the mRNA population than there are bands (Zimmerman et al., 1980). Some high res- olution genetic analyses also indicate the presence of more genes than bands in specific chromosome regions (Young and Judd, 1978; Zhimulev et al., 1981; Wright et al., 1981). In addition, several cloned Dro- sophila fragments have more than one coding region in lengths even shorter than the transcription units that we visualize-that is, three coding regions for heat shock proteins in 14 kb (Craig et al., 19791, four coding regions for larval cuticle proteins in 7.9 kb (Snyder et al., 19811, and four coding regions for chorion proteins in 12 or 18 kb (Spradling and Rubin, 1981). The above data are consistent with the possibility that the complex gradient fibril arrays are polycistronic transcription units that yield monocistronic messengers because of early cleavage.

Of course, we have no evidence that the observed complex gradient transcription units are involved in mRNA production. The exact nature of this early processing event will not likely come from further analysis of these transcription units whose identity cannot be established. We have initiated a similar electron microscopic project on a specific well defined transcript in which the cleavage and splicing sites for mRNA formation are known. That is, we have identified transcripts of the adenovirus major late transcription unit in chromatin spreads (Beyer et al., 19811, and we are in the process of hnRNP fibril analysis.

Experimental Procedures

Chromatin was dispersed for electron microscopic visualization from embryos and larval fat bodies of both D. melanogaster and C. erythrocephala. Embryos (2 to 4 hr old) were dechorionated by rolling on Scotch tape. Fat bodies quickly were dissected from third instar larvae in a few drops of pH 8.5 glass-distilled water. (This and all following “pH 8.5” solutions were adjusted to pH 8.5 with pH 10.00 buffer standard: potassium carbonate-potassium borate-potassium hydroxide buffer, 0.05 M. from Fischer Scientific.) Individual Dro- sophila embryos were transferred to a drop of 0.05% Joy detergent (pH 8.5) macerated with forceps and allowed to disperse for 5 min with occasional mixing by pipetting. A drop of 0.1 M sucrose in 10% formalin (pH 8.5) was added. After 2 to 5 more min, the preparation was transferred to a microcentrifugation chamber and centrifuged through a sucrose cushion onto an electron microscope grid. Addi- tional details of grid preparation have been described by Miller and Bakken, 1972 and McKnight and Miller, 1976. Drosophila fat bodies were subjected to the same procedure except that the dispersal time was 15 min. Calliphora embryos and fat bodies were macerated in a drop of solution containing 0.05% Joy, 0.1% digitonin and 50 pg/ml E. coli tRNA. pH 8.5 (M. Jamrich and 0. L. Miller, Jr., manuscript submitted). They were mixed by pipetting and a drop of 0.05 M sucrose (pH 8.5) was added. After 15 min dispersal, two drops of the sucrose-formalin solution were added. Fifteen min later, the preparations were transferred to the microcentrifugation chamber for deposition on the electron microscope grid.

Grids were stained with 1% phosphotungstic acid in 70% ethanol and were rotary-shadowed with platinum (Miller and Bakken, 1972). The following procedures have been described: microscopic examination and photography (k&Knight and Miller, 1979); identification and classification of transcription units as nucleolar or nonnucleolar

(Foe et al., 1976; McKnight and Miller, 1979); and contour length measuring (McKnight and Miller, 1979; Beyer et al., 1981).

Transcription units were subjected to linear regression analysis (Foe et al., 1976). That is, the length of each transcript was measured and graphed as a function of its DNA axial position with respect to the most recently initiated fibril of the array, and the best linear equation describing this distribution was fit by the least squares method. If a single line represents a good fit to this series of points (as determined by the value of the correlation coefficient), then it can be assumed that the transcripts initiated at a single site and have not been cleaved or broken while nascent (Foe et al., 1976). For the transcription units analyzed in this report, the data are fit significantly better by two or three linear equations (with similar slopes and different intercepts) than they are fit by a single linear equation (compare Laird and Chooi. 1976). The best-fit line to the first gradient of fibril length (that is, to the fibrils that presumably have not been cleaved) was used to estimate the transcription initiation site and to align RNA sequences in the RNP fibril maps (see text). Transcription unit lengths were estimated as the distance between the extrapolated initiation site and the most mature RNP fibril.

The conversion of contour length measurement (pm) to molecular size (kb) is based on the assumption that DNA contraction is inversely proportional to RNP fiber frequency and varies on a linear continuum between 3.3 kb/pm (the contraction ratio of fully transcribed rDNA in these preparations) and 4.8 kb/pm (the contraction ratio of nucleo- somal DNA in these preparations) (McKnight and Miller, 1979). The equation y = -0.049x + 4.87, where x is RNP fibers/pm DNP and y is DNA kb/gm. describes this relationship. Examination of the transcription units analyzed reveals that nucleosomes are indeed present between RNP fibrils on the sparsely transcribed genes.

The construction of RNP fibril maps has been described by Eeyer et al. (1980). The procedure used in this report varies somewhat because of the 5’ end cleavages (see text). Figure 1 describes how different RNP structural features are depicted on the RNP fibril maps.

Acknowledgments

We thank Milan Jamrich for generously sharing his micrographs of Calliphora nonribosomal transcription units. We thank Paul Adler, Kathy Martin, Linda Saffer and Ted Wright for helpful comments on the manuscript, and we acknowledge the financial support of the NIH to O.L.M.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

Received July 6, 1981

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correlation of hnrnp structure and nascent transcript cleavage

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